Tobias Tertel

Individual human immunodeficiency virus (HIV) particles can differ in their envelope protein (Env) despite being genetically identical. This non-genetic diversity can influence antibody recognition and has been implicated in the formation of persistent virus fractions that escape complete inhibition by broadly neutralizing antibodies. Investigating non-genetic diversity in HIV and other viruses requires approaches capable of characterizing antibody binding to individual viral particles. Flow virometry provides a promising tool for single-particle analysis, but a system combining high biosafety with specific particle labeling and versatility is lacking. Here, we developed a third-generation lentiviral vector system specifically designed for flow virometry. Fluorescent proteins were incorporated into the HIV-1 Gag polyprotein between the matrix- and capsid-encoding regions, flanked by viral protease cleavage sites, creating the packaging constructs pLentiFlow-eGFP and pLentiFlow-mCherry. Lentiviral particles (LVPs) based on pLentiFlow vectors were fluorescent and retained infectivity comparable to LVPs generated with the parental vector system despite reduced particle production. We optimized LVP detection on a CytoFLEX nano and defined a linear p24-based concentration range for reliable single-particle analysis. Using complementary controls, we demonstrated that background from the assessed sources accounted for no more than approximately 3% of events within a broad eGFP+ gate and confirmed that the detected fluorescent particles were predominantly membrane-enclosed. As a proof of concept, LVPs were pseudotyped with vesicular stomatitis virus glycoprotein, severe acute respiratory syndrome coronavirus 2 spike, or HIV-1 Env. Envelope-specific antibody binding was detected using 8G5F11, CR3022 or PGT128, respectively, demonstrating that different viral surface proteins can be analyzed on a common particle backbone. Together, these results establish a third-generation lentiviral vector system for flow virometry that combines intrinsic particle labeling with high biosafety and flexible pseudotyping. The system provides a platform for investigating envelope protein-associated non-genetic diversity and its consequences for antibody recognition in HIV and other viruses. Author summary: Recent studies suggest that non-genetic diversity in the envelope protein of the human immunodeficiency virus (HIV) influences antibody recognition, a phenomenon potentially relevant to other viruses as well. Understanding such non-genetic differences may aid the development of antibody-based vaccines and therapies. To assess envelope protein-associated non-genetic diversity within viral swarms, methods are needed to analyze individual viruses. Flow virometry is a promising approach but current methods face challenges in combining single-particle detection by flow cytometry with high biosafety and versatility. Here, we present a third-generation lentiviral vector system that generates fluorescent lentiviral particles (LVPs) for direct detection. These LVPs are produced from a reduced and split HIV genome and cannot produce infectious virus after infecting a cell, providing a high level of biosafety. The incorporated viral envelope proteins can be exchanged, allowing the study of HIV and other viruses. We characterize the LVPs, optimize their detection, establish conditions for reliable analysis of individual particles, and demonstrate the versatility of the system by detecting antibody binding to envelope proteins from HIV and two other viruses. By combining biosafety and versatility, this system broadens access to flow virometry and enables the study of non-genetic differences within swarms of HIV and other viruses.

Background: Owing to their potent immunomodulatory properties, mesenchymal stromal cell (MSC)-derived small extracellular vesicles (EVs) have emerged as promising neuroprotective treatments for ischemic stroke. Preclinical studies using MSC-EVs have mainly been performed in young, otherwise healthy rodents. Stroke patients frequently carry vascular risk factors and comorbidities. We herein investigated whether MSC-EVs retain neuroprotective activity in hyperlipidemic mice on cholesterol-rich Western diet. Methods: Male C57BL/6J mice were exposed to regular normal diet or Western diet for 6 weeks. At the age of 9-10 weeks, mice were exposed to transient intraluminal middle cerebral artery occlusion (MCAO). Vehicle or MSC-EVs (2x10 ⁶ or 6x10 ⁶ cell equivalents) were intravenously administered immediately after reperfusion, and vehicle or rosuvastatin (5 mg/kg/day) were intraperitoneally applied starting immediately after or seven days before MCAO. Neurological deficits, ischemic injury, and immune responses were evaluated up to 72 hours post-ischemia. To investigate the hyperlipidemia-associated immune dysregulation, mice received DNase-I before or immediately after MCAO. In defined subgroups, monocytes/ macrophages or neutrophils were additionally depleted by clodronate liposomes or anti-Ly6G antibodies, respectively. Results: In contrast to normolipidemic control mice, MSC-EVs failed to induce post-ischemic neuroprotection in hyperlipidemic mice. Neither MSC-EV dose escalation nor rosuvastatin co-treatment restored the therapeutic efficacy of MSC-EVs. Hyperlipidemia induced systemic innate immune dysregulation characterized by reduced monocyte/ macrophage activation, increased neutrophil activation, and elevated circulating cell-free DNA. DNase-I treatment before, but not after MCAO reversed these immune abnormalities and restored neuroprotection by MSC-EVs, decreasing neurological deficits, infarct volume and brain edema. Depletion of either monocytes/ macrophages or neutrophils abolished the neuroprotective effects of MSC-EVs in DNase-I-pretreated hyperlipidemic mice. Conclusions: Immune dysregulation abolishes MSC-EV-induced neuroprotection after ischemic stroke in hyperlipidemic mice. DNase-I priming restores MSC-EV responsiveness through mechanisms critically involving monocyte/ macrophage and neutrophil rebalancing. Our data highlight the host immune status as determinant of EV therapeutic efficacy.

The surface protein CD177 on human neutrophil granulocytes is linked to tissue infiltration. In most individuals, circulating neutrophils comprise distinct fractions of CD177‐expressing (CD177 ⁺ ) and non‐expressing (CD177 ⁻ ) cells, which differ functionally and influence disease outcomes. Currently, CD177 is considered a dynamic activation marker ultimately expressed by most neutrophils. Here, we show that, instead, CD177 ⁻ ‐neutrophils represent a stable subpopulation that never acquires CD177. CD177 ⁺ ‐ to CD177 ⁻ ‐neutrophil ratios persist over time in individuals, regardless of circadian rhythms or inflammation. Neutrophils remain CD177 ⁻ during in vitro stimulation and are morphologically similar to CD177 ⁺ ‐cells, but differ markedly regarding protein composition. Following stem cell transplantation, the host bone marrow defines the CD177‐pattern of mature neutrophils. Functionally, CD177 ⁻ neutrophils display pro‐tumoral properties, including elevated arginase expression and enhanced tumor‐supporting activity, are enriched in human head‐and‐neck cancer tissues, and associated with adverse clinical outcomes. Hence, CD177‐expression or ‐absence defines stable human neutrophil subtypes with distinct functions.

Imaging flow cytometry (IFCM) has emerged as a powerful method for high‐throughput phenotyping of small extracellular vesicles (sEVs) at single‐vesicle resolution. However, the reliability and sensitivity of IFCM critically depend on the performance of the antibodies used. In this study, we systematically compared several commercially available anti‐PD‐L1 antibodies for their ability to detect PD‐L1‐positive sEVs. Despite being derived from the same clone, antibodies from different manufacturers showed striking differences in labelling efficiency. These findings demonstrate that antibody validation for cellular targets does not ensure suitability for single‐EV analysis and underscore the need for rigorous, application‐specific qualification. To address this, we developed a robust IFCM workflow in full alignment with MIFlowCyt‐EV reporting standards. Using a qualified anti‐PD‐L1 antibody, we achieved reproducible detection of PD‐L1 ⁺ sEVs directly in unprocessed plasma samples from tumour patients. To assess the quantitative potential of single‐EV analysis, we compared this approach with bead‐based capture and performed dilution experiments. While bead‐based methods yielded a relatively narrow range of mean fluorescence intensities across samples, IFCM revealed distinct PD‐L1 ⁺ sEV levels, supporting its semi‐quantitative capability. Finally, we applied this workflow to plasma samples from patients with head and neck, lung, and breast cancer, as well as healthy donors. In subsets of all three cancer cohorts, elevated PD‐L1 ⁺ sEV levels were observed, suggesting potential clinical relevance. While in‐depth clinical correlations lie beyond the scope of this study, our findings establish a validated, standardized protocol for IFCM‐based single‐EV profiling and highlight the central role of antibody quality in ensuring analytical accuracy.

BACKGROUND: Neutrophil granulocytes actively contribute to tissue damage after ischemic stroke. The membrane protein CD (cluster of differentiation)177 is detectable on variable neutrophil numbers in most individuals (CD177 wild-type [CD177 ^WT ] genotype), whereas ≈5% of the general population completely lack CD177 (CD177-deficient [CD177 ^null ] genotype). Despite its known relevance in vasculitis, the role of ischemic stroke remains unknown. METHODS: In 2 prospective cohorts of patients with first-ever ischemic stroke (PROSCIS-B [Prospective Cohort With Incident Stroke Berlin], NOFF-S [Neutrophils: Origin, Fate & Function Stroke]), we assessed the effect of CD177 ^null and CD177 ^WT status on stroke severity and outcome (National Institutes of Health Stroke Scale and modified Rankin Scale) over 1 year or 3 months poststroke, respectively. By flow cytometry, we stratified CD177 expression level as CD177 ^neg , CD177 ^dim , and CD177 ^high . The predictive value of the CD177 state was evaluated by multivariable regression and discrimination analyses. RESULTS: In PROSCIS‐B (n=579; mean age, 68.1 years; 38.5% women) and NOFF-S (n=236, 68.4 years, 36.9% women), similar rates of patients were CD177 ^null (n=26 [4.5%] and n=10 [4.2%], respectively). Patients with CD177 ^null had a higher probability of unfavorable stroke outcome (modified Rankin Scale score 3–6) than patients with CD177 ^WT (n=8 of 21 [38.1%] versus 90 of 462 [19.5%] with follow-up, P =0.05, in PROSCIS-B; n=8 of 10 [80.0%] versus n=23 of 142 [16.2%] with follow-up, P P dim neutrophils at admission was negatively associated with stroke severity at admission, while that of CD177 ^high neutrophils predicted a favorable clinical outcome after 3 months. CD177 expression level significantly improved the prediction of stroke outcome in addition to clinical adjustment variables in area under the curve, net reclassification improvement, and integrated discrimination improvement analyses ( P =0.004, P =0.001, and P high ). CONCLUSIONS: CD177 expression at admission is an easy-to-measure biomarker for patient stratification. CD177 holds potential as a therapeutic target to modulate immune responses after stroke. REGISTRATION: URL: https://www.clinicaltrials.gov ; Unique identifier: NCT01363856. URL: https://drks.de/search/en/trial/Unique identifier: DRKS00030825.

Extracellular vesicles (EVs) are emerging as diagnostic and therapeutic agents, yet their nanoscale size limits quantitative single‐vesicle analysis of surface epitopes and molecular interactions. Flow‐Induced Dispersion Analysis (FIDA) is a microfluidic technique enabling real‐time measurement of hydrodynamic size and interaction kinetics using fluorescent ligands without requiring surface immobilization. Here, FIDA was applied to characterize EV–antibody interactions using anti‐CD63 antibodies across three EV sources: EVs from genetically engineered HEK cells expressing CD63‐eGFP or CD63‐NeonGreen‐Fc domain (CD63‐NG‐Fc), and EVs from clonally expanded immortalized mesenchymal stromal cells (ciMSCs). FIDA‐measured EV diameter sizes ranged from 40 to 90 nm, compared to 70–200 nm as determined by Nanoparticle Tracking Analysis (NTA), likely reflecting methodological differences or NTA's sensitivity to non‐EV particles. CD63‐binding EC ₅₀  values were 1.9 × 10 ⁸ particles/mL for CD63‐eGFP EVs and 8.4 × 10 ⁸ to 2.4 × 10 ⁹ particles/mL for ciMSC EVs, indicating higher epitope abundance in engineered vesicles. Within a broader antibody titration, maximal antibody loading to the Fc‐receptor domain on EVs was observed at 0.5–5 nM, reaching 30%–40% for IgG1 and 15%–20% for Cetuximab. These results establish FIDA as a high‐resolution, label‐efficient tool for quantifying EV‐antibody interactions and epitope accessibility, supporting its integration into EV standardization and quality control workflows.

Impaired bone healing is a major challenge in orthopedic and trauma surgery, often causing long-term disability and high costs. While autologous bone grafting is the gold standard, it is limited by donor site morbidity, low availability, and surgical risks. As an alternative, surgical site-released tissue (SSRT) collected intraoperatively offers a readily available source of regenerative cells and bioactive factors. This study investigates the potential of SSRT-derived mesenchymal stromal cell (MSC)-like cells and their extracellular vesicles (EVs) to support bone healing in a cell-free approach. SSRT samples from 30 patients undergoing elective hip replacement were collected using a surgical vacuum filter. MSC-like cells were isolated and characterized based on International Society for Cellular Therapy (ISCT) criteria. Interestingly, many SSRT-derived MSC-like cells expressed CD34, a marker typically absent in cultured MSCs but linked to tissue-resident stromal cells, suggesting distinct regenerative properties. These cells also showed slow proliferation rates (P1: 8.7 ± 3.2 days; P2: 8.2 ± 5.4 days). EVs were isolated from osteogenically stimulated (EVs^MSC/O+) and unstimulated (EVs^MSC/O−) MSCs over three weeks. Antibody profiling revealed distinct cargo compositions, with a notable enrichment of CD13+ EVs in the stimulated group. Further in vivo and functional studies are needed to clarify underlying mechanisms and confirm therapeutic efficacy.

Extracellular vesicles derived from human induced mesenchymal stromal cells (hiEVs) constitute a promising cell-free therapeutic option for osteoarthritis.

Efficient phagocytosis of pathogens is a key effector function of the innate immune system. Impaired phagocytic activity can result in uncontrolled pathogen proliferation and life-threatening infections. However, reliable methods to detect early dysfunction of the phagocytic system in vivo are limited. Here, we used a mouse model of Listeria monocytogenes infection to determine blood parameters which correlate with limited macrophage function. We found that lack of macrophages led to accumulation of nuclei in the blood. Further analysis of nuclei revealed that these nuclei were released from bone marrow-derived cells. Macrophage-depleted mice and interferon-gamma-deficient mice, which are known to have reduced phagocytotic capacity, showed increased amounts of free nuclei. This was associated with lethal outcome and occurrence of acute hepatopathy in these mice after Listeria monocytogenes infection. Our findings highlight a simple and noninvasive method to assess macrophage phagocytic function in vivo , which should be assessed in further murine and human studies as a tool for predicting host vulnerability to infection.