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. 2025 Oct 9;19(41):36027–36034. doi: 10.1021/acsnano.5c07444

Extracellular Vesicles and B Cell Interactions: B‑Side Track or Greatest Hit?

Zach Troyer †, Olesia Gololobova †, Tom A P Driedonks ‡, Loren D Erickson §,∥, Joy Wolfram ⊥,#,*, Kenneth W Witwer †,¶,∇,*
PMCID: PMC12548337  PMID: 41067713

Abstract

Substantial efforts have been made in understanding interactions between extracellular vesicles and cells, particularly in small animal models, such as rats and mice. However, many questions remain unanswered about the fate of intravenously injected extracellular vesicles in large animals, especially in terms of interactions with immune cells. In this article, we summarize findings from two recent nonhuman primate studies involving extracellular vesicle interactions with circulating immune cells. We highlight the surprising finding that certain extracellular vesicles primarily associate with B cells and present key questions for future studies pertaining to this interaction.

Keywords: exosomes, immune cells, intravenous administration, microvesicles, peripheral blood mononuclear cells, phagocytosis

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1. Introduction

Several small animal studies have evaluated extracellular vesicle (EV) interactions with cells and tissues following systemic administration. − However, few studies have assessed the fate of EVs in large animal models, which more closely recapitulate human physiology. The natural capacity of EVs to communicate between cells has led to much interest in using them for therapeutic purposes. For therapeutic and drug delivery applications, it is especially important to understand how EVs interact with immune cells in large animal models, as immunological recognition can impede or accelerate therapeutic effects. Uncovering EV interactions in large animal models is also critical for developing new diagnostic tools and understanding EV-mediated effects in health and disease.

EVs used in therapeutic studies have been administered through a variety of routes, with intravenous injection being the most common. , Systemically administered EVs are exposed to the body’s natural circulating immune system, encountering innate and adaptive immune cells in addition to soluble factors released by these cells. In vivo studies have demonstrated that nonhost EVs are rapidly cleared from the blood by liver phagocytes and have a short half-life, especially after repeated administrations due to an antibody-mediated phenomenon termed accelerated blood clearance (ABC). , Immediately upon entering the bloodstream, the EV surface obtains a “corona” of host biomolecules, through tenuous surface interactions. − EVs then encounter a variety of circulating host immune cells: T cells, B cells, natural killer cells, monocytes, macrophages, dendritic cells, basophils, eosinophils, and neutrophils, as well as platelets and red blood cells. Monocyte lineage cells (dendritic cells, macrophages), which encompass the body’s major phagocytic cells, play an important role in immune surveillance in the blood and are primarily responsible for engulfing and breaking down nanostructures, including cellular debris, cancerous/damaged cells, pathogens, and foreign nanoparticles. ,

In this article, we report and reflect upon the surprising finding that B cells primarily associate with fluorescent EVs in nonhuman primate blood of the pigtailed macaque (Macaca nemestrina) both ex vivo and in vivo. B cells associate with fluorescent EVs regardless of the source of the EVs and isolation method. , This finding is unexpected, given that B cells are less abundant in circulation compared to other immune cells, and are not typically known for phagocytosis, which is a process more commonly associated with cells like macrophages, neutrophils, and dendritic cells. Although phagocytosis is suspected, the mechanistic nature of this association remains unclear. EVs can interact with cells through a variety of pathways, including surface binding, endocytosis/uptake, and, in some cases, direct fusion with the cellular plasma membrane. While the receptor-mediated endocytosis pathway used by antigen-specific B cells to internalize small antigens is relatively well understood, there is limited evidence documenting the ability of B cells to engage in phagocytosis of particles larger than antigens or membrane fusion with EVs. − Evidence has emerged demonstrating that an evolutionarily conserved B cell subset in mice, B1 cells, have phagocytic capabilities, particularly involving bacteria. , Similarly, a human B cell line was shown to be capable of phagocytosing Mycobacterium tuberculosis and latex beads. Another study demonstrated that human B cells could phagocytose soluble antigens and large latex beads in a manner independent from conventionally defined endocytosis pathways and dependent on Ras homologue gene family, member G (RhoG). Importantly, B cells from primitive vertebrates were shown to have potent phagocytic capabilities, suggesting that ancestral B cells may have been more phagocytic and evolved from a phagocytic cell type. Taken together, these studies raise intriguing questions about the broader functional capabilities of B cells in immune responses and suggest potential roles beyond their classical functions in antigen presentation and antibody production. In light of the literature and data from our studies, we are of the opinion that key questions concerning B cell/EV interactions should be investigated in the future.

2. B Cell Biogenesis and Function

B cells are an essential constituent of the vertebrate humoral adaptive immune system, producing antibodies that facilitate recognition and clearance of foreign material. Originating from the bone marrow after a selection process to remove those that recognize self-antigens, B cells travel through the blood to reach the spleen and mature into naïve B cells. Naïve B cells predominantly reside in the spleen and lymph nodes, where they encounter antigens present in the blood and lymphatic fluid, respectively. Upon recognition of a specific antigen by the B cell receptor (BCR), and typically with the help of antigen-specific CD4+ T cells, B cells undergo activation, leading to extensive proliferation. This activation induces B cells to differentiate either into antibody-producing plasma cells or into memory B cells, which remain poised for rapid response upon subsequent encounters with the same antigen. , Plasma cells often migrate from the lymphoid organs to the bone marrow, where they can continue producing antibodies, while memory B cells can circulate throughout the body for years, providing long-term immunity. As a result, the blood of a healthy individual is expected to contain a heterogeneous population of naïve and memory B cells, with plasma cells primarily residing in the bone marrow. Both naïve and memory B cells are recognized as professional antigen-presenting cells, owing to their critical roles in antigen recognition and presentation to T cells. The B cell receptor (BCR) binds to a specific antigen, initiating receptor-mediated endocytosis, subsequent proteolytic processing of the antigen, and its presentation on major histocompatibility complex II (MHC-II) molecules to T cells.

3. Extracellular Vesicles Associate Primarily with B Cells in Non-Human Primate Blood

In a prior study examining the pharmacokinetics and biodistribution of EVs, EVs derived from Expi293F cells (HEK293 embryonic kidney cell origin) transfected with a plasmid encoding the EV-tracking molecule PalmGRET were injected intravenously into pigtailed macaques (Figure A). Blood was drawn at 1, 3, 5, and 10 min post-EV administration. Peripheral blood mononuclear cells (PBMCs) and granulocytes within the blood were then analyzed for association with EVs, as indicated by the presence of EV-luminal enhanced green fluorescent protein (EGFP), originating from the PalmGRET molecule. EV interactions with various PBMC subtypes and granulocytes were detectable as early as 1 min postadministration. Surprisingly, up to 81% of B cells were EGFP positive, a substantially higher proportion than observed in other cell populations, which displayed less than 15% EGFP positivity (Figure B). Even after adjusting for the relative abundance of these cells in circulation, B cells were still the largest EGFP-positive population (Figure B, pie charts). Surprisingly, only a small portion of monocytes associated with EVs. When the spleen was assessed 1 h after EV administration, the majority of EGFP-positive splenic cells were also B cells. The EVs used in this case, were derived from human cells, which may have led to their recognition as foreign in the primate bloodstream and could have further impacts on the B cell association.

1.

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Comparison of B cell/extracellular vesicle (EV) association over time in vivo and ex vivo models. (A) Schematic of the experimental workflow. For the in vivo study, EVs were injected intravenously into pigtailed macaques. Blood was collected for measurement at 1, 3, 5, and 10 min postadministration. For the ex vivo study, EVs were mixed with blood from pigtailed macaque donors on a temperature-controlled rotator. Measurements were performed at 60, 120, 240, and 1440 min post-EV introduction. (B,C) Line graphs show the percentage of various blood cells that associated with EVs at different time points in the two models (B: in vivo, C: ex vivo). The dotted line at 18% is the maximum EV/cell association seen for any non-B-cell cell type. The pie charts show cell type prevalence-adjusted EV association values for the 1 min (in vivo) or 60 min (ex vivo) time points: EGFP positivity adjusted by the prevalence of each cell type in the PBMC population (B cells = 9%, T cells = 61%, monocytes = 5%). Other measured PBMCs and granulocytes displayed negligible EV interactions (not shown). Data are reused from refs and and combined with previously unpublished data (first time point for ex vivo study). Partially created with BioRender.com.

3.1. Extracellular Vesicles from Various Sources Associate with B Cells Ex Vivo

Following up on the surprising B cell findings from the in vivo studies, an ex vivo assay was developed to assess EV interactions with whole blood. In this assay, EVs were mixed with fresh whole blood from pigtailed macaque donors and incubated at 37 °C on a mixer to simulate a dynamic environment (Figure A). Matching the in vivo results (intravenously injected EVs, Figure B), ∼80% of B cells in the ex vivo assay were EGFP-positive (Figure C). B cell association was observed at the first (40 min) ex vivo time point and decayed slightly over a 24 h period (Figure C). The dynamics of EV/B cell interactions varied in vivo compared with ex vivo. The decay of EV signal associated with B cells was much faster in vivo, likely due to the effects of active circulation and clearance organs (for example, liver). , Consistent with the in vivo findings, B cells were the most common EGFP-positive PBMC subtype at all time points measured, despite accounting for only ∼9% of the total PBMCs (Figure B, pie charts). To determine whether the observed B cell associations were specific to the EV type (Expi293F cell-derived) or the isolation method (tangential flow filtration combined with size-exclusion chromatography), EVs were also produced from U87 MG glioma cells using two different isolation techniques: ultracentrifugation and size-exclusion chromatography. Notably, EVs from U87 MG cells, isolated by both techniques, predominantly interacted with B cells in the ex vivo assays, mirroring the results observed with Expi293F-derived EVs. When the dosage of Expi293F-derived EVs was increased, the predominant EGFP-positive population shifted to monocytes when accounting for cell type abundance in the blood (of note, this donor exhibited an atypically high monocyte-to-B cell ratio), although B cells still displayed the highest population positivity (∼88% versus 26% in monocytes). In the in vivo experiments, repeated intravenous administration of EVs eventually elicited immunoglobulin G (IgG) antibodies in plasma that were directed against the administered EVs. , Theoretically, these anti-EV-IgGs could bind to Fc receptors on B cells or other cells, thereby facilitating association of EVs. However, ex vivo experiments with whole blood from naïve subjects showed a comparably high association with B cells, ruling out a determinative contribution of antibody-mediated association.

4. Other EV Studies with Animal Models

Various in vivo studies in mice have tracked the distribution of EVs following intravenous injection. − , These studies have focused on organ distribution and association with specific target cells, demonstrating that EVs primarily accumulate in the liver and spleen. However, only a few studies have assessed to what extent EVs associate with circulating immune cells. ,,

Wiklander et al. investigated the distribution of human and mouse EVs derived from various cell types and administered intravenously in a mouse model. Based on organ/tissue-level in vivo imaging (IVIS) detection of HEK293T EVs labeled with the fluorescent lipophilic dye, DiR, approximately 60% and 15% of the total fluorescent signal was found in liver and spleen, respectively. Mouse B16F10 melanoma EVs had a similar profile. In contrast, EVs from mouse C2C12 muscle cells had enhanced liver accumulation, while mouse primary bone marrow dendritic cell EVs had reduced liver and higher spleen accumulation.

Grange et al. introduced human mesenchymal stem cell (MSC) EVs intravenously into mice with acute kidney injury (AKI) and imaged with IVIS. EVs were labeled with fluorescent lipophilic dyes postproduction (DL–EVs) or produced from membrane-labeled cells (LCD–EVs). DL–EVs had greater kidney association than LCD–EVs in the AKI model. Healthy mice did not have detectable EV accumulation in the kidney, suggesting an important role of kidney injury in biodistribution. However, the major organs taking up EVs in both AKI and healthy mice were spleen and liver.

Lai et al. injected HEK293T EVs modified to carry Gaussia princeps luciferase intravenously into mouse models via the retroorbital vein. Per IVIS imaging of bioluminescence, EV accumulation was detected primarily in liver and spleen, similar to other studies. By follow-up analysis of dissected organs, EV signal was highest in the spleen, followed by liver, lungs, and kidney. Other tissues (brain, heart, muscle) had low but detectable levels of EV signal. Interestingly, when organs were perfused to reduce signal from blood EVs, the kidney had the highest EV signal, while spleen dropped to the lowest of all organs tested.

Lázaro-Ibáñez et al. compared the biodistribution of Expi293F EVs labeled by different methods in mice to assess potential labeling method biases. DiR-labeled EVs resulted in signal almost exclusively localized to liver and spleen, with fluorescent signal detected by IVIS. EVs labeled with a radioactive indium compound ([111In]-DTPA) to allow detection by single-photon emission computed tomography (SPECT) coupled with computed tomography (CT) also had strong liver and spleen signal, with smaller amounts in the kidneys. In contrast, EVs labeled with nanoluciferase (Nluc) produced the greatest signal in the lungs, followed by spleen and liver.

Pham et al. studied the cellular biodistribution of fluorescent lipid dye Aco-490 labeled EVs from human red blood cells (RBCEVs) injected into mice, after previously finding that RBCEVs predominately accumulated in liver and spleen. , In the liver, RBCEVs signal was associated primarily with Kupffer cells. In the spleen, signal was found mostly in or on CD169+ cells (likely macrophages or other monocyte-lineage cells). In human PBMC populations exposed to RBCEVs, monocytes had the highest levels of association, followed by B cells, NK cells, and T cells.

Taken together, several studies have assessed organ distribution of EVs in mice, while only one looked at circulating cells. In the case of RBCEVs in mice, B cells exhibited the second highest level of interactions among circulating cells. Further studies, including with multiple EV types, are needed to determine whether B cell interactions with EVs are more prominent in primates than in mice.

5. Potential Factors Mediating EV Association with B Cells

Our finding that EVs associate primarily with primate B cells compared with other circulating cells , was obtained with EVs obtained from two sources and separated with different methods. This finding is unexpected for the following reasons: (i) B cells are not known to have large-scale phagocytic functions and (ii) monocytes, macrophages and dendritic cells are the primary phagocytic cells in blood. It also raises additional questions, including whether EVs associate with B cells through surface interactions, cellular internalization, or direct membrane fusion; which EV and B cell biomolecules or other mediators facilitate interactions; and whether these associations serve any particular purpose (Figure , upper panel).

2.

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Unknowns of B cell/EV interactions. Upper panel: potential EV-B cell interaction modes include surface interactions (with or without leading to signal transduction) and internalization leading to endosomal escape or endosomal degradation. Middle panel: candidate surface molecules on the EV and B cell membranes that may mediate B cell/EV interactions. The EV corona may also play a critical role in these interactions. Indicated proteins are proposed examples and are neither suggestive of specific protein–protein interactions nor meant to exclude other interactions. Lower panel: B cell subsets that may interact with EVs (currently unknown). Created with BioRender.com.

Elements of the EV biomolecular corona may contribute to EV-cell association (Figure middle panel). Upon intravenous administration, EVs quickly accumulate a corona of blood components. − , Extensive proteomic characterization of EVs isolated from or spiked into blood, along with careful controls to remove nonadhered contaminants, has revealed the identities of common EV corona proteins. These include apolipoproteins such as apolipoprotein (apo)­A-I and apoE, albumin, clusterins, cytochrome oxidases, fibrinogens, and immunoglobulins. Both EV corona factors and native EV proteins may be contribute to B cell tropism.

Studies in which known integral EV surface proteins are removed or preblocked could aid in determining if these proteins play a role in mediating B cell association. One strategy is utilizing genetic knockouts of proteins like CD9, CD63, CD81, or other integral EV membrane proteins in EV-producing cells and compare B cell association of these EVs with those from wild type cells. Alternatively, EVs could be incubated with antibodies against membrane proteins prior to introduction into the blood, possibly blocking specific protein–protein interactions. EVs could also be treated with a low concentration of trypsin to digest membrane proteins present at the EV surface, although this would not reveal the identity of the exact proteins critical for B cell interaction.

Assessing the potential impact of the EV corona on B cell interactions is challenging if EVs are introduced into whole blood, as a corona will form simultaneously with B cell interactions. As a workaround, B cells can be purified from whole blood and then mixed with EVs independent of the soluble biomolecules present in plasma. Levels of EV-B cell interaction could also be compared for EVs that were or were not preincubated with plasma. Additionally, different EV separation methods (e.g., ultracentrifugation, size-exclusion chromatography, ultrafiltration), can be used to strip the corona to different degrees: for example, size-exclusion chromatography and ultracentrifugation are reported to remove parts of the EV corona, while tangential flow filtration preserves it. These techniques could be used to determine whether EV corona biomolecules that may mediate EV/B cell interactions exist in the soft (loosely attached) or hard (strongly bound) EV corona.

Considering the wide range of potential interaction mediators, it is also important to assess the degree to which different B cell subtypes interact with EVs. Our studies assessed CD20+ B cells, which includes all B cell subtypes except terminally differentiated plasma cells (Figure bottom panel). Moreover, EV/B cell interactions took place despite the vigorous fluidic movement of the bloodstream, suggesting specific and strong interactions. The dynamics of the interactions between EVs and B cells may shed further light on the strength of the EV/B cell association. Time course measurements revealed that B cell association was immediate and then slowly decreased over time. ,

6. Potential Functions of EV/B Cell Interactions and Immunological Context

It is well established that EVs can impact immune responses, serving in their role as intercellular communicators. Much of this work stems from the field of T cell biology, where EVs were shown to induce and inhibit T cell activation, dependent on the producer cell and the EV cargo. − EVs from cancer cells have a propensity to induce T cell senescence and create a premetastatic niche, while EVs derived from dendritic cells can induce T cell activation through display of costimulatory molecules and delivery of antigens. , Acting through a separate mechanism, EVs can also transfer surface receptors from one T cell to another, creating a newly signal-susceptible T cell population.

Given their complex role in the immune system, EVs could have similar impacts on B cells, modulating their functions as a means of immunological control. EVs derived from T cells could carry the T cell receptor and costimulatory molecules required for T cell help and canonical B cell activation, inducing this process through surface contact with B cells in the absence of direct cell contact. EVs from dendritic cells or other cells could deliver antigens directly to B cells, bypassing the traditional antigen phagocytosis route. More generally, EVs could alter the activation threshold of B cells through signaling cascades, making them more or less likely to be activated, depending on the EV cell of origin and internal or surface cargo. Downstream of activation, EVs could influence trafficking of B cells to the germinal centers of lymphoid follicles, where they normally differentiate into memory B cells or antibody-secreting plasma cells. Additionally, EVs might manipulate the propensity of B cells to undergo class switching and somatic hypermutation into a particular antibody subclass (for example, IgG versus IgM). These speculated EV impacts on B cells would require experimental evidence but are plausible based on known EV functions.

7. Outlook and Conclusion

Outside the EV field, there is evidence of synthetic nanoparticles preferentially interacting with B cells, potentially through phagocytosis. In 2012, it was shown that B cells could engulf and phagocytose both latex microspheres (500–2000 nm diameter) and complement-opsonized E. coli. , Following up on these findings in 2016, Tsoi et al. demonstrated that B cells were partially responsible for uptake of quantum dots in the liver after in vivo administration. Interestingly, over 80% of B cells from the 2016 study were quantum dot-positive at 12 h postadministration. Most surprisingly, the levels of B cell uptake were comparable to Kupffer cells, the liver’s dedicated macrophages, despite making up a small percentage of the liver cells. Taken together, our studies on EVs and studies on synthetic nanoparticles suggest that there is an underexplored function of B cells pertinent to the nanomedicine and EV fields. Future studies are necessary to better understand B cell/EV interactions. An understanding of the mechanisms driving these interactions would allow improvements in therapeutic platforms, whether by exploitation of B cell tropism to deliver payloads directly to B cells, or by controlled avoidance of these cells after intravenous administration. Reflecting on the data discussed in this article, we hope that it spurs investigation into unanswered/underexplored questions (Box 1).7.

Acknowledgments

The in vivo and ex vivo studies were both supported by the U.S. National Institutes of Health (NIH) through AI144997. The Witwer laboratory has also been supported in part by the NIH under Awards DA047807, MH118164, and CA241694; the Paul G. Allen Frontiers Foundation; the Michael J. Fox Foundation (Grant 00900821); and the Richman Family Precision Medicine Center of Excellence in Alzheimer’s Disease at Johns Hopkins University. This work was partially funded by Ionis Pharmaceuticals under the Ion-ARPA Operation Payload Delivery scheme (K.W.W., J.W.) and The University of Queensland, Australia (J.W.). J.W. was also supported by the National Heart Foundation of Australia under Award 108500-2024_FLF, the Medical Research Future Fund, Australia under Award MRF2019485, the National Breast Cancer Foundation, Australia under Award 2023/IIRS0063, and the National Institute on Aging, NIH under Award R01AG076537. We also acknowledge all the authors of the original in vivo and ex vivo studies for their important contributions. The content is solely the responsibility of the authors and does not necessarily represent the official views of the organizations and funding agencies. BioRender was used to create some of the figures.

Conceptualization: ZT, JW Data curation: ZT Formal analysis: ZT Funding acquisition: ZT, OG, JW, KWW Investigation: OG, TAPD Methodology: OG, TAPD, KWW Resources: OG, TAPD, KWW Visualization: ZT Writingoriginal draft: ZT Writingreview and editing: ZT, OG, TAPD, LDE, JW, KWW.

The authors declare the following competing financial interest(s): K.W.W. is or has been an advisory board member of B4 RNA, Exopharm, NeuroDex, NovaDip, ReNeuron, and ShiftBio; holds stock options with NeuroDex; and privately consults as Kenneth Witwer Consulting. Ionis Pharmaceuticals, Yuvan Research, and AgriSciX have sponsored or are sponsoring research in the Witwer laboratory. J.W. is or has been a board member or scientific advisor of Omnidermal, Genomill, and Pharmatest Services. Ionis Pharmaceuticals and Sartorius have sponsored or are sponsoring research in the Wolfram laboratory.

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