Investigation of exosomal tetraspanin profile in sepsis patients as a promising diagnostic biomarker.

INTRODUCTION: Sepsis, a leading cause of mortality globally, has a complex and multifaceted pathophysiology which still requires elucidation. Therefore, this study aimed to analyze and quantify the number of exosomes in sepsis patients from a South African cohort using the ExoView (NanoView Biosciences, Boston, MA) platform. METHODS: Blood samples were collected from black South African patients attending the local Intensive Care Unit (ICU) hospital. Exosomes were isolated and characterize via TEM and CD63 ELISA kits. ExoView was used to determine particle count, particle size distribution and colocalization of different tetraspanin markers. RESULTS: Exosomal levels in sepsis patients were significantly higher compared to the control group (p < 0.05). Sepsis exosomes showed a homogenous size distribution ranging from 55 to 70 nm. Tetraspanin colocalization analysis revealed that sepsis exosomes have significantly higher CD63/CD9, CD63/CD81 and CD63/CD9/CD81 colocalization percentages than the control group. CONCLUSION: This unique tetraspanin colocalization pattern of sepsis exosomes could serve as a potential sepsis biomarker. Further investigations are required to identify sepsis exosomal cargo signatures for further understanding of sepsis pathophysiology in order to develop effective diagnostics and treatments.

Membrane organization by tetraspanins and galectins shapes lymphocyte function.

Immune receptors are not randomly distributed at the plasma membrane of lymphocytes but are segregated into specialized domains that function as platforms to initiate signalling, as exemplified by the B cell or T cell receptor complex and the immunological synapse. 'Membrane-organizing proteins' and, in particular, tetraspanins and galectins, are crucial for controlling the spatiotemporal organization of immune receptors and other signalling proteins. Deficiencies in specific tetraspanins and galectins result in impaired immune synapse formation, lymphocyte proliferation, antibody production and migration, which can lead to impaired immunity, tumour development and autoimmunity. In contrast to conventional ligand-receptor interactions, membrane organizers interact in cis (on the same cell) and modulate receptor clustering, receptor dynamics and intracellular signalling. New findings have uncovered their complex and dynamic nature, revealing shared binding partners and collaborative activity in determining the composition of membrane domains. Therefore, immune receptors should not be envisaged as independent entities and instead should be studied in the context of their spatial organization in the lymphocyte membrane. We advocate for a novel approach to study lymphocyte function by globally analysing the role of membrane organizers in the assembly of different membrane complexes and discuss opportunities to develop therapeutic approaches that act via the modulation of membrane organization.

Extracellular vesicle distribution and localization in skeletal muscle at rest and following disuse atrophy.

BACKGROUND: Skeletal muscle (SkM) is a large, secretory organ that produces and releases myokines that can have autocrine, paracrine, and endocrine effects. Whether extracellular vesicles (EVs) also play a role in the SkM adaptive response and ability to communicate with other tissues is not well understood. The purpose of this study was to investigate EV biogenesis factors, marker expression, and localization across cell types in the skeletal muscle. We also aimed to investigate whether EV concentrations are altered by disuse atrophy. METHODS: To identify the potential markers of SkM-derived EVs, EVs were isolated from rat serum using density gradient ultracentrifugation, followed by fluorescence correlation spectroscopy measurements or qPCR. Single-cell RNA sequencing (scRNA-seq) data from rat SkM were analyzed to assess the EV biogenesis factor expression, and cellular localization of tetraspanins was investigated by immunohistochemistry. Finally, to assess the effects of mechanical unloading on EV expression in vivo, EV concentrations were measured in the serum by nanoparticle tracking analysis in both a rat and human model of disuse. RESULTS: In this study, we show that the widely used markers of SkM-derived EVs, α-sarcoglycan and miR-1, are undetectable in serum EVs. We also found that EV biogenesis factors, including the tetraspanins CD63, CD9, and CD81, are expressed by a variety of cell types in SkM. SkM sections showed very low detection of CD63, CD9, and CD81 in myofibers and instead accumulation within the interstitial space. Furthermore, although there were no differences in serum EV concentrations following hindlimb suspension in rats, serum EV concentrations were elevated in human subjects after bed rest. CONCLUSIONS: Our findings provide insight into the distribution and localization of EVs in SkM and demonstrate the importance of methodological guidelines in SkM EV research.

Extracellular vesicles engineered to bind albumin demonstrate extended circulation time and lymph node accumulation in mouse models.

Extracellular vesicles (EVs) have shown promise as potential therapeutics for the treatment of various diseases. However, their rapid clearance after administration could be a limitation in certain therapeutic settings. To solve this, an engineering strategy is employed to decorate albumin onto the surface of the EVs through surface display of albumin binding domains (ABDs). ABDs were either included in the extracellular loops of select EV-enriched tetraspanins (CD63, CD9 and CD81) or directly fused to the extracellular terminal of single transmembrane EV-sorting domains, such as Lamp2B. These engineered EVs exert robust binding capacity to human serum albumins (HSA) in vitro and mouse serum albumins (MSA) after injection in mice. By binding to MSA, circulating time of EVs dramatically increases after different routes of injection in different strains of mice. Moreover, these engineered EVs show considerable lymph node (LN) and solid tumour accumulation, which can be utilized when using EVs for immunomodulation, cancer- and/or immunotherapy. The increased circulation time of EVs may also be important when combined with tissue-specific targeting ligands and could provide significant benefit for their therapeutic use in a variety of disease indications.

Tetraspanins distinguish separate extracellular vesicle subpopulations in human serum and plasma - Contributions of platelet extracellular vesicles in plasma samples.

BACKGROUND: The ability to isolate extracellular vesicles (EVs) from blood is vital in the development of EVs as disease biomarkers. Both serum and plasma can be used, but few studies have compared these sources in terms of the type of EVs that are obtained. The aim of this study was to determine the presence of different subpopulations of EVs in plasma and serum. METHOD: Blood was collected from healthy subjects, and plasma and serum were isolated in parallel. ACD or EDTA tubes were used for the collection of plasma, while serum was obtained in clot activator tubes. EVs were isolated utilising a combination of density cushion and SEC, a combination of density cushion and gradient or by a bead antibody capturing system (anti-CD63, anti-CD9 and anti-CD81 beads). The subpopulations of EVs were analysed by NTA, Western blot, SP-IRIS, conventional and nano flow cytometry, magnetic bead ELISA and mass spectrometry. Additionally, different isolation protocols for plasma were compared to determine the contribution of residual platelets in the analysis. RESULTS: This study shows that a higher number of CD9+ EVs were present in EDTA-plasma compared to ACD-plasma and to serum, and the presence of CD41a on these EVs suggests that they were released from platelets. Furthermore, only a very small number of EVs in blood were double-positive for CD63 and CD81. The CD63+ EVs were enriched in serum, while CD81+ vesicles were the rarest subpopulation in both plasma and serum. Additionally, EDTA-plasma contained more residual platelets than ACD-plasma and serum, and two centrifugation steps were crucial to reduce the number of platelets in plasma prior to EV isolation. CONCLUSION: These results show that human blood contains multiple subpopulations of EVs that carry different tetraspanins. Blood sampling methods, including the use of anti-coagulants and choice of centrifugation protocols, can affect EV analyses and should always be reported in detail.

Kinetics and interaction studies of anti-tetraspanin antibodies and ICAM-1 with extracellular vesicle subpopulations using continuous flow quartz crystal microbalance biosensor.

Continuous flow quartz crystal microbalance (QCM) was utilized to study binding kinetics between EV subpopulations (exomere- and exosome-sized EVs) and four affinity ligands: monoclonal antibodies against tetraspanins (anti-CD9, anti-CD63, and anti-CD81) and recombinant intercellular adhesion molecule-1 (ICAM-1) or CD54 protein). High purity CD9+, CD63+, and CD81+ EV subpopulations of <50 nm exomeres and 50-80 nm exosomes were isolated and fractionated using our recently developed on-line coupled immunoaffinity chromatography - asymmetric flow field-flow fractionation system. Adaptive Interaction Distribution Algorithm (AIDA), specifically designed for the analysis of complex biological interactions, was used with a four-step procedure for reliable estimation of the degree of heterogeneity in rate constant distributions. Interactions between exomere-sized EVs and anti-tetraspanin antibodies demonstrated two interaction sites with comparable binding kinetics and estimated dissociation constants Kd ranging from nM to fM. Exomeres exhibited slightly higher affinity compared to exosomes. The highest affinity with anti-tetraspanin antibodies was achieved with CD63+ EVs. The interaction of EV subpopulations with ICAM-1 involved in cell internalization of EVs was also investigated. EV - ICAM-1 interaction was also of high affinity (nM to pM range) with overall lower affinity compared to the interactions of anti-tetraspanin antibodies and EVs. Our findings proved that QCM is a valuable label-free tool for kinetic studies with limited sample concentration, and that advanced algorithms, such as AIDA, are crucial for proper determination of kinetic heterogeneity. To the best of our knowledge, this is the first kinetic study on the interaction between plasma-derived EV subpopulations and anti-tetraspanin antibodies and ICAM-1.

Imaging of surface microdomains on individual extracellular vesicles in 3-D.

Extracellular vesicles (EVs) are secreted from all cell types and are intimately involved in tissue homeostasis. They are being explored as vaccine and gene therapy platforms, as well as potential biomarkers. As their size is below the diffraction limit of light microscopy, direct visualizations have been daunting and single-particle studies under physiological conditions have been hampered. Here, direct stochastic optical reconstruction microscopy (dSTORM) was employed to visualize EVs in three-dimensions and to localize molecule clusters such as the tetraspanins CD81 and CD9 on the surface of individual EVs. These studies demonstrate the existence of membrane microdomains on EVs. These were confirmed by Cryo-EM. Individual particle visualization provided insights into the heterogeneity, structure, and complexity of EVs not previously appreciated.

Framework for rapid comparison of extracellular vesicle isolation methods.

Extracellular vesicles (EVs) are released by all cells into biofluids and hold great promise as reservoirs of disease biomarkers. One of the main challenges in studying EVs is a lack of methods to quantify EVs that are sensitive enough and can differentiate EVs from similarly sized lipoproteins and protein aggregates. We demonstrate the use of ultrasensitive, single-molecule array (Simoa) assays for the quantification of EVs using three widely expressed transmembrane proteins: the tetraspanins CD9, CD63, and CD81. Using Simoa to measure these three EV markers, as well as albumin to measure protein contamination, we were able to compare the relative efficiency and purity of several commonly used EV isolation methods in plasma and cerebrospinal fluid (CSF): ultracentrifugation, precipitation, and size exclusion chromatography (SEC). We further used these assays, all on one platform, to improve SEC isolation from plasma and CSF. Our results highlight the utility of quantifying EV proteins using Simoa and provide a rapid framework for comparing and improving EV isolation methods from biofluids.

CD9 inhibition reveals a functional connection of extracellular vesicle secretion with mitophagy in melanoma cells.

Tetraspanins are often used as Extracellular Vesicle (EV) detection markers because of their abundance on these secreted vesicles. However, data on their function on EV biogenesis are controversial and compensatory mechanisms often occur upon gene deletion. To overcome this handicap, we have compared the effects of tetraspanin CD9 gene deletion with those elicited by cytopermeable peptides with blocking properties against tetraspanin CD9. Both CD9 peptide or gene deletion reduced the number of early endosomes. CD9 peptide induced an increase in lysosome numbers, while CD9 deletion augmented the number of MVB and EV secretion, probably because of compensatory CD63 expression upregulation. In vivo, CD9 peptide delayed primary tumour cell growth and reduced metastasis size. These effects on cell proliferation were shown to be concomitant with an impairment in mitochondrial quality control. CD9 KO cells were able to compensate the mitochondrial malfunction by increasing total mitochondrial mass reducing mitophagy. Our data thus provide the first evidence for a functional connection of tetraspanin CD9 with mitophagy in melanoma cells.

High-Throughput Counting and Superresolution Mapping of Tetraspanins on Exosomes Using a Single-Molecule Sensitive Flow Technique and Transistor-like Semiconducting Polymer Dots.

A method for high-throughput counting and superresolution mapping of surface proteins on exosomes is described. The method combines a single-molecule sensitive flow technique and an adaptive superresolution imaging method. Exosomes stained with membrane dye and dye-conjugated antibodies were analyzed using a microfluidic platform at a flow rate of 100 exosome s-1 to determine size and protein copy number. Superresolution mapping was performed with exosomes labeled with novel transistor-like, semiconducting polymer dots (Pdots), which exhibit spontaneous blinking with <5 nm localization error and a broad range of optical-adjustable duty cycles. Based on the copy numbers extracted from the flow analysis, the switch-on frequency of the Pdots were finely adjusted so that structures of hundreds of exosomes were obtained within five minutes. The high throughput and high sensitivity of this method offer clear advantages for characterization of exosomes and similar biological vesicles.

Extracellular Vesicles Analysis in the COVID-19 Era: Insights on Serum Inactivation Protocols towards Downstream Isolation and Analysis.

Since the outbreak of the COVID-19 crisis, the handling of biological samples from confirmed or suspected SARS-CoV-2-positive individuals demanded the use of inactivation protocols to ensure laboratory operators' safety. While not standardized, these practices can be roughly divided into two categories, namely heat inactivation and solvent-detergent treatments. These routine procedures should also apply to samples intended for Extracellular Vesicles (EVs) analysis. Assessing the impact of virus-inactivating pre-treatments is therefore of pivotal importance, given the well-known variability introduced by different pre-analytical steps on downstream EVs isolation and analysis. Arguably, shared guidelines on inactivation protocols tailored to best address EVs-specific requirements will be needed among the analytical community, yet deep investigations in this direction have not yet been reported. We here provide insights into SARS-CoV-2 inactivation practices to be adopted prior to serum EVs analysis by comparing solvent/detergent treatment vs. heat inactivation. Our analysis entails the evaluation of EVs recovery and purity along with biochemical, biophysical and biomolecular profiling by means of a set of complementary analytical techniques: Nanoparticle Tracking Analysis, Western Blotting, Atomic Force Microscopy, miRNA content (digital droplet PCR) and tetraspanin assessment by microarrays. Our data suggest an increase in ultracentrifugation (UC) recovery following heat treatment; however, it is accompanied by a marked enrichment in EVs-associated contaminants. On the other hand, solvent/detergent treatment is promising for small EVs (<150 nm range), yet a depletion of larger vesicular entities was detected. This work represents a first step towards the identification of optimal serum inactivation protocols targeted to EVs analysis.

Extracellular vesicles isolated from porcine seminal plasma exhibit different tetraspanin expression profiles.

Seminal extracellular vesicles (EVs) include exosomes (ø 40-120 nm) and microvesicles (MVs, ø 120-1000 nm), which would be involved in multiple functional reproductive roles. The study aimed to establish which EV subtypes are present in pig semen, using a high-resolution flow cytometer to explore differences in their tetraspanin expression profile. The EVs were isolated from 12 pig ejaculates using serial ultracentrifugation and characterized by dynamic light scattering and electron microscopy for size and morphology as well as for tetraspanin expression using flow cytometry with Carboxyfluorescein succinimidyl ester (CFSE) and antibodies against CD9, CD63 and CD81. Pig semen contained a heterogeneous EV-population regarding size and morphology. Flow cytometric analysis demonstrated that the proportion of EVs expressing CD63 and CD9 was higher in MVs (P < 0.001 and P < 0.05, respectively) than in exosomes, while the opposite was true for CD81; higher (P < 0.001) in exosomes than in MVs. In conclusion, (1) the new generation of flow cytometers are able to accurately identify EVs and to gate them in two size-different populations named exosomes and MVs. (2) Tetraspanins CD9, CD63 and CD81 are present in both seminal EVs, albeit with exosomes and MVs differing in expression profiles, suggesting dissimilar cargo and binding affinity.

A Nanoparticle-Based Approach for the Detection of Extracellular Vesicles.

The analysis of extracellular vesicles (EVs) typically requires tedious and time-consuming isolation process from bio-fluids. We developed a nanoparticle-based time resolved fluorescence immunoassay (NP-TRFIA) that uses biotinylated antibodies against the proteins of tetraspanin family and tumor-associated antigens for capturing EVs from urine samples and cell culture supernatants without the need for isolation. The captured-EVs were detected either with Eu3+-chelate or Eu3+-doped nanoparticle-based labels conjugated either to antibodies against the tetraspanins or lectins targeting the glycan moieties on EVs surface. The NP-TRFIA demonstrated specific capturing and detection of EVs by antibodies and lectins. Lectin-nanoparticle based assays showed 2-10 fold higher signal-to-background ratio compared with lectin-chelate assays. The nanoparticle assay concept allowed surface glycosylation profiling of the urine derived-EVs with lectins. It was also applied to establish an assay showing differential expression of tumor-associated proteins on more aggressive (higher ITGA3 on DU145- and PC3-EVs) compared to less aggressive (higher EpCAM on LNCaP-EVs) PCa- cell lines derived-EVs. This NP-TRFIA can be used as a simple tool for analysis and characterization of EVs in urine and cell culture supernatants. Such approach could be useful in identification of disease-specific markers on the surface of patient-derived urinary EVs.

Anti-CD37 chimeric antigen receptor T cells are active against B- and T-cell lymphomas.

Chimeric antigen receptor (CAR) T cells have emerged as a novel form of treatment of patients with B-cell malignancies. In particular, anti-CD19 CAR T-cell therapy has effected impressive clinical responses in B-cell acute lymphoblastic leukemia and diffuse large B-cell lymphoma. However, not all patients respond, and relapse with antigen loss has been observed in all patient subsets. Here, we report on the design and optimization of a novel CAR directed to the surface antigen CD37, which is expressed in B-cell non-Hodgkin lymphomas, in chronic lymphocytic leukemia, and in some cases of cutaneous and peripheral T-cell lymphomas. We found that CAR-37 T cells demonstrated antigen-specific activation, cytokine production, and cytotoxic activity in models of B- and T-cell lymphomas in vitro and in vivo, including patient-derived xenografts. Taken together, these results are the first showing that T cells expressing anti-CD37 CAR have substantial activity against 2 different lymphoid lineages, without evidence of significant T-cell fratricide. Furthermore, anti-CD37 CARs were readily combined with anti-CD19 CARs to generate dual-specific CAR T cells capable of recognizing CD19 and CD37 alone or in combination. Our findings indicate that CD37-CAR T cells represent a novel therapeutic agent for the treatment of patients with CD37-expressing lymphoid malignancies.

Concentration-Normalized Electroanalytical Assaying of Exosomal Markers.

Exosomes are both active in mediating intracellular communication and potentially present a potent cargo of disease biomarkers to an assay. The robust evaluation of exosomal markers could lead to a paradigm shift in clinical analysis and associated care. To date, much of this has been hindered by issues of sample preparation and assay signal-to-noise. We introduce here the use of ultrasensitive electrochemical impedance spectroscopy to quantify both external (tetraspanin) and internal (syntenin) exosome-specific markers. Associated exosome detection limits are 1.9 × 105 particles mL-1 (equivalent to 320 aM or 9500 exosomes in 50 µL) for intact exosomes and 3-5 picomolar for internal exosomal syntenin levels with almost 5 decades of linear dynamic range. Sample preparation can be carried out by simple fine filtering of cell-conditioned medium prior to a non-NTA-determined (i.e., nanoparticle tracking analysis) exosome concentration analysis, lysing, and subsequent internal syntenin quantification. Such concentration-normalized dual-marker analysis can be used to define "analytical zones" in a manner which is then independent of absolute exosome concentration and sample preparation.

Therapeutic targeting of tetraspanin8 in epithelial ovarian cancer invasion and metastasis.

Epithelial ovarian cancer (EOC) invasion and metastasis are complex phenomena that result from the coordinated action of many metastatic regulators and must be overcome to improve clinical outcomes for patients with these cancers. The identification of novel therapeutic targets is critical because of the limited success of current treatment regimens, particularly in advanced-stage ovarian cancers. In this study, we found that tetraspanin 8 (TSPAN8) is overexpressed in about 52% (14/27) of EOC tissues and correlates with poor survival. Using small interfering RNA-mediated TSPAN8 knockdown and a competition assay with purified TSPAN8 large extracellular loop (TSPAN8-LEL) protein, we identified TSPAN8-LEL as a key regulator of EOC cell invasion. Furthermore, monotherapy with TSPAN8-blocking antibody we developed shows that antibody-based modulation of TSPAN8-LEL can significantly reduce the incidence of EOC metastasis without severe toxicity in vivo. Finally, we demonstrated that the TSPAN8-blocking antibody promotes the internalization and concomitant downregulation of cell surface TSPAN8. Collectively, our data suggest TSPAN8 as a potential novel therapeutic target in EOCs and antibody targeting of TSPAN8 as an effective strategy for inhibiting invasion and metastasis of TSPAN8-expressing EOCs.

Wiskott-Aldrich Syndrome Interacting Protein Deficiency Uncovers the Role of the Co-receptor CD19 as a Generic Hub for PI3 Kinase Signaling in B Cells.

Humans with Wiskott-Aldrich syndrome display a progressive immunological disorder associated with compromised Wiskott-Aldrich Syndrome Interacting Protein (WIP) function. Mice deficient in WIP recapitulate such an immunodeficiency that has been attributed to T cell dysfunction; however, any contribution of B cells is as yet undefined. Here we have shown that WIP deficiency resulted in defects in B cell homing, chemotaxis, survival, and differentiation, ultimately leading to diminished germinal center formation and antibody production. Furthermore, in the absence of WIP, several receptors, namely the BCR, BAFFR, CXCR4, CXCR5, CD40, and TLR4, were impaired in promoting CD19 co-receptor activation and subsequent PI3 kinase (PI3K) signaling. The underlying mechanism was due to a distortion in the actin and tetraspanin networks that lead to altered CD19 cell surface dynamics. In conclusion, our findings suggest that, by regulating the cortical actin cytoskeleton, WIP influences the function of CD19 as a general hub for PI3K signaling.

Multispectral imaging reveals the tissue distribution of tetraspanins in human lymphoid organs.

Multispectral imaging is a novel microscopy technique that combines imaging with spectroscopy to obtain both quantitative expression data and tissue distribution of different cellular markers. Tetraspanins CD37 and CD53 are four-transmembrane proteins involved in cellular and humoral immune responses. However, comprehensive immunohistochemical analyses of CD37 and CD53 in human lymphoid organs have not been performed so far. We investigated CD37 and CD53 protein expression on primary human immune cell subsets in blood and in primary and secondary lymphoid organs. Both tetraspanins were prominently expressed on antigen-presenting cells, with highest expression of CD37 on B lymphocytes. Analysis of subcellular distribution showed presence of both tetraspanins on the plasma membrane and on endosomes. In addition, CD53 was also present on lysosomes. Quantitative analysis of expression and localization of CD37 and CD53 on lymphocytes within lymphoid tissues by multispectral imaging revealed high expression of both tetraspanins on CD20(+) cells in B cell follicles in human spleen and appendix. CD3(+) T cells within splenic T cell zones expressed lower levels of CD37 and CD53 compared to T cells in the red pulp of human spleen. B cells in human bone marrow highly expressed CD37, whereas the expression of CD53 was low. In conclusion, we demonstrate differential expression of CD37 and CD53 on primary human immune cells, their subcellular localization and their quantitative distribution in human lymphoid organs. This study provides a solid basis for better insight into the function of tetraspanins in the human immune response.

The C-terminal tail of tetraspanin proteins regulates their intracellular distribution in the parasite Trichomonas vaginalis.

The parasite Trichomonas vaginalis is the causative agent of trichomoniasis, a prevalent sexually transmitted infection. Here, we report the cellular analysis of T.vaginalis tetraspanin family (TvTSPs). This family of membrane proteins has been implicated in cell adhesion, migration and proliferation in vertebrates. We found that the expression of several members of the family is up-regulated upon contact with vaginal ectocervical cells. We demonstrate that most TvTSPs are localized on the surface and intracellular vesicles and that the C-terminal intracellular tails of surface TvTSPs are necessary for proper localization. Analyses of full-length TvTSP8 and a mutant that lacks the C-terminal tail indicates that surface-localized TvTSP8 is involved in parasite aggregation, suggesting a role for this protein in parasite : parasite interaction.

Placental Hofbauer cells assemble and sequester HIV-1 in tetraspanin-positive compartments that are accessible to broadly neutralizing antibodies.

INTRODUCTION: Within monocyte-derived macrophages, HIV-1 accumulates in intracellular virus-containing compartments (VCCs) that are inaccessible to the external environment, which implicate these cells as latently infected HIV-1 reservoirs. During mother-to-child transmission of HIV-1, human placental macrophages (Hofbauer cells (HCs)) are viral targets, and have been shown to be infected in vivo and sustain low levels of viral replication in vitro; however, the risk of in utero transmission is less than 7%. The role of these primary macrophages as viral reservoirs is largely undefined. The objective of this study is to define potential sites of viral assembly, accumulation and neutralization in HCs given the pivotal role of the placenta in preventing HIV-1 infection in the mother-infant dyad. METHODS: Term placentae from 20 HIV-1 seronegative women were obtained following caesarian section. VCCs were evaluated by 3D confocal and electron microscopy. Colocalization R values (Pearson's correlation) were quantified with colocalization module of Volocity 5.2.1. Replication kinetics and neutralization studies were evaluated using p24 ELISA. RESULTS: We demonstrate that primary HCs assemble and sequester HIV-1(BaL) in intracellular VCCs, which are enriched in endosomal/lysosomal markers, including CD9, CD81, CD63 and LAMP-1. Following infection, we observed HIV-1 accumulation in potentially acidic compartments, which stained intensely with Lysotracker-Red. Remarkably, these compartments are readily accessible via the cell surface and can be targeted by exogenously applied small molecules and HIV-1-specific broadly neutralizing antibodies. In addition, broadly neutralizing antibodies (4E10 and VRC01) limited viral replication by HIV-1-infected HCs, which may be mediated by FcγRI. CONCLUSIONS: These findings suggest that placental HCs possess intrinsic adaptations facilitating unique sequestration of HIV-1, and may serve as a protective viral reservoir to permit viral neutralization and/or antiretroviral drug entry in utero.