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<front>
<journal-meta>
<journal-id journal-id-type="publisher-id">Front. Immunol.</journal-id>
<journal-title>Frontiers in Immunology</journal-title>
<abbrev-journal-title abbrev-type="pubmed">Front. Immunol.</abbrev-journal-title>
<issn pub-type="epub">1664-3224</issn>
<publisher>
<publisher-name>Frontiers Media S.A.</publisher-name>
</publisher>
</journal-meta>
<article-meta>
<article-id pub-id-type="doi">10.3389/fimmu.2024.1354065</article-id>
<article-categories>
<subj-group subj-group-type="heading">
<subject>Immunology</subject>
<subj-group>
<subject>Original Research</subject>
</subj-group>
</subj-group>
</article-categories>
<title-group>
<article-title>Immunoregulatory molecule expression on extracellular microvesicles in people living with HIV</article-title>
</title-group>
<contrib-group>
<contrib contrib-type="author">
<name>
<surname>Neyrinck-Leglantier</surname><given-names>Deborah</given-names>
</name>
<xref ref-type="aff" rid="aff1"><sup>1</sup></xref>
<xref ref-type="aff" rid="aff2"><sup>2</sup></xref>
<xref ref-type="aff" rid="aff3"><sup>3</sup></xref>
<uri xlink:href="https://loop.frontiersin.org/people/1425443"/>
<role content-type="https://credit.niso.org/contributor-roles/formal-analysis/"/>
<role content-type="https://credit.niso.org/contributor-roles/investigation/"/>
<role content-type="https://credit.niso.org/contributor-roles/writing-original-draft/"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Tamagne</surname><given-names>Marie</given-names>
</name>
<xref ref-type="aff" rid="aff1"><sup>1</sup></xref>
<xref ref-type="aff" rid="aff2"><sup>2</sup></xref>
<xref ref-type="aff" rid="aff3"><sup>3</sup></xref>
<uri xlink:href="https://loop.frontiersin.org/people/2046407"/>
<role content-type="https://credit.niso.org/contributor-roles/writing-review-editing/"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Ben Rayana</surname><given-names>Raida</given-names>
</name>
<xref ref-type="aff" rid="aff4"><sup>4</sup></xref>
<uri xlink:href="https://loop.frontiersin.org/people/2602526"/>
<role content-type="https://credit.niso.org/contributor-roles/writing-review-editing/"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Many</surname><given-names>Souganya</given-names>
</name>
<xref ref-type="aff" rid="aff5"><sup>5</sup></xref>
<role content-type="https://credit.niso.org/contributor-roles/writing-review-editing/"/>
<role content-type="https://credit.niso.org/contributor-roles/investigation/"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Vingert</surname><given-names>Paul</given-names>
</name>
<xref ref-type="aff" rid="aff1"><sup>1</sup></xref>
<xref ref-type="aff" rid="aff2"><sup>2</sup></xref>
<xref ref-type="aff" rid="aff3"><sup>3</sup></xref>
<role content-type="https://credit.niso.org/contributor-roles/writing-review-editing/"/>
<role content-type="https://credit.niso.org/contributor-roles/investigation/"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname>LeGagneux</surname><given-names>Julie</given-names>
</name>
<xref ref-type="aff" rid="aff1"><sup>1</sup></xref>
<xref ref-type="aff" rid="aff2"><sup>2</sup></xref>
<xref ref-type="aff" rid="aff3"><sup>3</sup></xref>
<role content-type="https://credit.niso.org/contributor-roles/writing-review-editing/"/>
<role content-type="https://credit.niso.org/contributor-roles/investigation/"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Delorme</surname><given-names>Ad&#xe8;le Silane</given-names>
</name>
<xref ref-type="aff" rid="aff1"><sup>1</sup></xref>
<xref ref-type="aff" rid="aff2"><sup>2</sup></xref>
<xref ref-type="aff" rid="aff3"><sup>3</sup></xref>
<role content-type="https://credit.niso.org/contributor-roles/writing-review-editing/"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Andrieu</surname><given-names>Muriel</given-names>
</name>
<xref ref-type="aff" rid="aff5"><sup>5</sup></xref>
<uri xlink:href="https://loop.frontiersin.org/people/989917"/>
<role content-type="https://credit.niso.org/contributor-roles/writing-review-editing/"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Boilard</surname><given-names>Eric</given-names>
</name>
<xref ref-type="aff" rid="aff6"><sup>6</sup></xref>
<xref ref-type="aff" rid="aff7"><sup>7</sup></xref>
<uri xlink:href="https://loop.frontiersin.org/people/321309"/>
<role content-type="https://credit.niso.org/contributor-roles/writing-review-editing/"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Cognasse</surname><given-names>Fabrice</given-names>
</name>
<xref ref-type="aff" rid="aff8"><sup>8</sup></xref>
<xref ref-type="aff" rid="aff9"><sup>9</sup></xref>
<uri xlink:href="https://loop.frontiersin.org/people/149514"/>
<role content-type="https://credit.niso.org/contributor-roles/writing-review-editing/"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Hamzeh-Cognasse</surname><given-names>Hind</given-names>
</name>
<xref ref-type="aff" rid="aff9"><sup>9</sup></xref>
<uri xlink:href="https://loop.frontiersin.org/people/171230"/>
<role content-type="https://credit.niso.org/contributor-roles/writing-review-editing/"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Perez-Patrigeon</surname><given-names>Santiago</given-names>
</name>
<xref ref-type="aff" rid="aff10"><sup>10</sup></xref>
<uri xlink:href="https://loop.frontiersin.org/people/1691242"/>
<role content-type="https://credit.niso.org/contributor-roles/writing-review-editing/"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Lelievre</surname><given-names>Jean-Daniel</given-names>
</name>
<xref ref-type="aff" rid="aff4"><sup>4</sup></xref>
<uri xlink:href="https://loop.frontiersin.org/people/1191245"/>
<role content-type="https://credit.niso.org/contributor-roles/writing-review-editing/"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Pirenne</surname><given-names>France</given-names>
</name>
<xref ref-type="aff" rid="aff1"><sup>1</sup></xref>
<xref ref-type="aff" rid="aff2"><sup>2</sup></xref>
<xref ref-type="aff" rid="aff3"><sup>3</sup></xref>
<uri xlink:href="https://loop.frontiersin.org/people/1599900"/>
<role content-type="https://credit.niso.org/contributor-roles/writing-review-editing/"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Gallien</surname><given-names>S&#xe9;bastien</given-names>
</name>
<xref ref-type="aff" rid="aff4"><sup>4</sup></xref>
<role content-type="https://credit.niso.org/contributor-roles/writing-review-editing/"/>
</contrib>
<contrib contrib-type="author" corresp="yes">
<name>
<surname>Vingert</surname><given-names>Beno&#xee;t</given-names>
</name>
<xref ref-type="aff" rid="aff1"><sup>1</sup></xref>
<xref ref-type="aff" rid="aff2"><sup>2</sup></xref>
<xref ref-type="aff" rid="aff3"><sup>3</sup></xref>
<xref ref-type="author-notes" rid="fn001"><sup>*</sup></xref>
<uri xlink:href="https://loop.frontiersin.org/people/1855534"/>
<role content-type="https://credit.niso.org/contributor-roles/conceptualization/"/>
<role content-type="https://credit.niso.org/contributor-roles/supervision/"/>
<role content-type="https://credit.niso.org/contributor-roles/validation/"/>
<role content-type="https://credit.niso.org/contributor-roles/writing-original-draft/"/>
</contrib>
</contrib-group>
<aff id="aff1"><sup>1</sup><institution>Univ Paris Est-Creteil (UPEC), Institut National de la Sant&#xe9; et de la Recherche M&#xe9;dicale (INSERM), Institut Mondor de la Recherche Biom&#xe9;dicale (IMRB)</institution>, <addr-line>Creteil</addr-line>, <country>France</country></aff>
<aff id="aff2"><sup>2</sup><institution>Etablissement Fran&#xe7;ais du Sang (EFS)</institution>, <addr-line>Ivry-sur-Seine</addr-line>, <country>France</country></aff>
<aff id="aff3"><sup>3</sup><institution>Laboratory of Excellence, Biog&#xe9;n&#xe8;se et Pathologies du Globule Rouge (GR-Ex)</institution>, <addr-line>Paris</addr-line>, <country>France</country></aff>
<aff id="aff4"><sup>4</sup><institution>Service de Maladies Infectieuses et Immunologie Clinique, Centre Hospitalier Universitaire Henri-Mondor, Assistance Publique-H&#xf4;pitaux de Paris (AP-HP), Universit&#xe9; Paris-Est Cr&#xe9;teil (UPEC)</institution>, <addr-line>Cr&#xe9;teil</addr-line>, <country>France</country></aff>
<aff id="aff5"><sup>5</sup><institution>Institut Cochin, Inserm U1016, Centre National de la Recherche Scientifique (CNRS) UMR8104, Universit&#xe9; Paris-Cit&#xe9;</institution>, <addr-line>Paris</addr-line>, <country>France</country></aff>
<aff id="aff6"><sup>6</sup><institution>Facult&#xe9; de M&#xe9;decine and Centre de Recherche ARThrite, Universit&#xe9; Laval</institution>, <addr-line>Qu&#xe9;bec, QC</addr-line>, <country>Canada</country></aff>
<aff id="aff7"><sup>7</sup><institution>Centre de Recherche du Centre Hospitalier Universitaire de Qu&#xe9;bec, Universit&#xe9; Laval</institution>, <addr-line>Qu&#xe9;bec, QC</addr-line>, <country>Canada</country></aff>
<aff id="aff8"><sup>8</sup><institution>Etablissement Fran&#xe7;ais du Sang Auvergne-Rh&#xf4;ne-Alpes</institution>, <addr-line>Saint-Etienne</addr-line>, <country>France</country></aff>
<aff id="aff9"><sup>9</sup><institution>SAINBIOSE, INSERM, U1059, University of Lyon</institution>, <addr-line>Saint-Etienne</addr-line>, <country>France</country></aff>
<aff id="aff10"><sup>10</sup><institution>Division of Infectious Diseases, Queen&#x2019;s University</institution>, <addr-line>Kingston, ON</addr-line>, <country>Canada</country></aff>
<author-notes>
<fn fn-type="edited-by">
<p>Edited by: Mario Clerici, University of Milan, Italy</p>
</fn>
<fn fn-type="edited-by">
<p>Reviewed by: Caroline Subra, Henry M. Jackson Foundation for the Advancement of Military Medicine (HJF), United States</p>
<p>Aikaterini Alexaki, Centre Hospitalier Universitaire Vaudois (CHUV), Switzerland</p>
</fn>
<fn fn-type="corresp" id="fn001">
<p>*Correspondence: Beno&#xee;t Vingert, <email xlink:href="mailto:benoit.vingert@inserm.fr">benoit.vingert@inserm.fr</email>
</p>
</fn>
</author-notes>
<pub-date pub-type="epub">
<day>04</day>
<month>03</month>
<year>2024</year>
</pub-date>
<pub-date pub-type="collection">
<year>2024</year>
</pub-date>
<volume>15</volume>
<elocation-id>1354065</elocation-id>
<history>
<date date-type="received">
<day>11</day>
<month>12</month>
<year>2023</year>
</date>
<date date-type="accepted">
<day>20</day>
<month>02</month>
<year>2024</year>
</date>
</history>
<permissions>
<copyright-statement>Copyright &#xa9; 2024 Neyrinck-Leglantier, Tamagne, Ben Rayana, Many, Vingert, LeGagneux, Delorme, Andrieu, Boilard, Cognasse, Hamzeh-Cognasse, Perez-Patrigeon, Lelievre, Pirenne, Gallien and Vingert</copyright-statement>
<copyright-year>2024</copyright-year>
<copyright-holder>Neyrinck-Leglantier, Tamagne, Ben Rayana, Many, Vingert, LeGagneux, Delorme, Andrieu, Boilard, Cognasse, Hamzeh-Cognasse, Perez-Patrigeon, Lelievre, Pirenne, Gallien and Vingert</copyright-holder>
<license xlink:href="http://creativecommons.org/licenses/by/4.0/">
<p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</p>
</license>
</permissions>
<abstract>
<sec>
<title>Introduction</title>
<p>People living with HIV (PLWH) now benefit from combined antiviral treatments that durably control viral replication. These antiretroviral treatments decrease mortality and improve quality of life in PLWH, but do not completely control the excessive non-specific activation of the immune system in PLWH. This chronic immune activation is a key element of HIV immunopathology that contributes to the pathophysiology of inflammatory comorbid conditions, such as cardiovascular disorders, cancer and autoimmune diseases. Circulating non-exosomal extracellular vesicles, also known as microparticles (MPs) are detected in these diseases and have been linked to immune activation. The objective of this study was to characterize the MPs present in PLWH and to assess their association with chronic immune activation.</p>
</sec>
<sec>
<title>Methods</title>
<p>We performed flow cytometry for the complete phenotypic characterization of MPs from fresh plasma from PLWH and from people without HIV as the control group. The absolute number, size and cellular origin of MPs were evaluated. The immunoregulatory profile was determined by cell origin, for MPs derived from platelets (PMPs), monocytes (MMPs) and T lymphocytes (LMPs).</p>
</sec>
<sec>
<title>Results</title>
<p>PLWH had significantly more circulating MPs than controls, for MPs of all sizes originating from T lymphocytes, red blood cells, neutrophils, dendritic cells, B lymphocytes and endothelial cells. PMPs and MMPs were not more numerous in PLWH, but the immunoregulatory phenotypes of these MPs differed between PLWH and controls. These differences in immunoregulatory molecule expression profile were also observed for LMPs. PDL1, ICOSL, CCR5, TGF&#x3b2;1, MHC classes I and II, TRAIL, CXCR4, OX40, DC-SIGN, CTLA4 and PDL2 were more strongly expressed on the surface of MPs from PLWH than on those from controls.</p>
</sec>
<sec>
<title>Conclusion</title>
<p>MPs are an important element in intercellular communication, making it possible to transfer phenotypes and functions to immune cells. The significantly higher numbers of MPs expressing diverse immunomodulatory molecules in PLWH may make a major contribution to the maintenance and/or the development of immune-cell activation in these individuals.</p>
</sec>
</abstract>
<kwd-group>
<kwd>immune activation (IA)</kwd>
<kwd>extracellular vesicle (EV)</kwd>
<kwd>people living with HIV (PLWH)</kwd>
<kwd>microparticles (MPs)</kwd>
<kwd>immunoregulatory molecules</kwd>
<kwd>chronic immune activation</kwd>
</kwd-group>
<counts>
<fig-count count="2"/>
<table-count count="0"/>
<equation-count count="0"/>
<ref-count count="39"/>
<page-count count="8"/>
<word-count count="4454"/>
</counts>
<custom-meta-wrap>
<custom-meta>
<meta-name>section-in-acceptance</meta-name>
<meta-value>Viral Immunology</meta-value>
</custom-meta>
</custom-meta-wrap>
</article-meta>
</front>
<body>
<sec id="s1" sec-type="intro">
<title>Introduction</title>
<p>Antiretroviral treatment (ART) controls viral replication in people living with HIV (PLWH), thereby increasing life expectancy, but successful treatment requires high levels of adherence and ART can have multiple adverse effects in long-term use. PLWH on ART have a higher life expectancy due to a decrease in the incidence of opportunistic diseases associated with immune recovery, but they may experience other non-infectious diseases, such as cardiovascular diseases and cancer, which may be favored by excessive non-specific chronic activation of the immune system. This chronic activation contributes an immune aging process, constitutes a significant barrier to the development of immune therapy and is a key factor in the immunopathology of HIV (<xref ref-type="bibr" rid="B1">1</xref>).</p>
<p>The extracellular vesicles (EVs) secreted by cells can be classified into several subgroups, including exosomes, apoptotic bodies, and non-exosomal extracellular vesicles, which are also known as microparticles (MPs), microvesicles, or ectosomes (<xref ref-type="bibr" rid="B2">2</xref>&#x2013;<xref ref-type="bibr" rid="B4">4</xref>). MPs are generally larger (200-900 nm) than exosomes and are generated by budding of the plasma membrane (<xref ref-type="bibr" rid="B5">5</xref>). These vesicles are present in the blood and carry membrane and cytoplasmic elements, such as membrane receptors, cytokines and nucleic acids, from the cell of origin (<xref ref-type="bibr" rid="B5">5</xref>&#x2013;<xref ref-type="bibr" rid="B7">7</xref>).</p>
<p>MPs are involved in intercellular communication and can modulate the immune system through interactions with many immune cells (conventional CD4<sup>+</sup> TL, Tfh, Th17, Treg, monocytes, B lymphocytes, dendritic cells) (<xref ref-type="bibr" rid="B8">8</xref>&#x2013;<xref ref-type="bibr" rid="B15">15</xref>). The underlying mechanisms remain incompletely understood, but these interactions may involve immune ligands/receptors present on the surface of the MPs. We recently showed that CD27<sup>+</sup> and CD70<sup>+</sup> MPs can transfer these receptors to CD4<sup>+</sup> TLs, thereby increasing activation and lymphoproliferation (<xref ref-type="bibr" rid="B11">11</xref>).</p>
<p>MPs are also linked to the pathogenesis of viral infections, through roles in viral dissemination, inflammation, and modulation of the immune system (<xref ref-type="bibr" rid="B12">12</xref>, <xref ref-type="bibr" rid="B16">16</xref>, <xref ref-type="bibr" rid="B17">17</xref>). EVs have been shown to play an important role in the diffusion of HIV infection via the transfer of coreceptors, notably CCR5 and CXCR4 (<xref ref-type="bibr" rid="B18">18</xref>, <xref ref-type="bibr" rid="B19">19</xref>). They may also reduce HIV-associated humoral responses by inhibiting the <italic>in vitro</italic> production of immunoglobulins G and A by memory B cells (<xref ref-type="bibr" rid="B20">20</xref>). However, almost all studies on EVs and HIV have focused exclusively on the total population of EVs (<xref ref-type="bibr" rid="B21">21</xref>&#x2013;<xref ref-type="bibr" rid="B23">23</xref>). As a result, it is not possible to draw any firm conclusions on the role of a specific subset of EVs, such as MPs, in HIV infection and the modulation of immune responses. Nevertheless, we know that the concentration of circulating MPs increases in PLWH (<xref ref-type="bibr" rid="B20">20</xref>, <xref ref-type="bibr" rid="B24">24</xref>&#x2013;<xref ref-type="bibr" rid="B26">26</xref>) and that the MP phenotype may be altered in PLWH, which may have an even greater impact on the activation state of immune cells and the regulation of their response.</p>
<p>We used flow cytometry for extensive characterization of the phenotypes of MPs from fresh plasma obtained from PLWH, comparing the results for these individuals with those for people without HIV. We determined the absolute number, size, and cellular origin of MPs. We also characterized the proteins expressed on the membrane of these MPs with a panel of cellular activation and immune checkpoint markers potentially involved in chronic immune cell activation. The immunoregulatory profile of MPs by cell origin was also specified for those with the strongest immunoregulatory potential: MPs derived from platelets (PMPs), monocytes (MMPs) and T lymphocytes (LMPs) (<xref ref-type="bibr" rid="B8">8</xref>, <xref ref-type="bibr" rid="B9">9</xref>, <xref ref-type="bibr" rid="B15">15</xref>, <xref ref-type="bibr" rid="B27">27</xref>, <xref ref-type="bibr" rid="B28">28</xref>).</p>
</sec>
<sec id="s2" sec-type="materials|methods">
<title>Materials and methods</title>
<sec id="s2_1">
<title>Study population</title>
<p>We used fresh blood samples from patients followed in the infectious diseases and clinical immunology department of Henri-Mondor University Hospital (Creteil, France). The patients included had at least five years of follow-up for HIV infection. They were on active antiretroviral therapy, had an undetectable viral load (&lt; 50 copies/mL) and had a CD4/CD8 ratio greater than 0.5. Demographics, clinical and treatment characteristics, and virological outcomes were collected. Healthy blood donors were enrolled as a control group for comparison with the PLWH, with whom they were matched for sex, ethnicity, and body mass index (BMI) (<xref ref-type="supplementary-material" rid="SM1"><bold>Supplementary Tables S1</bold></xref>, <xref ref-type="supplementary-material" rid="SM1"><bold>S2</bold></xref>). Blood samples from the control group were provided by the French national blood bank (<italic>Etablissement Fran&#xe7;ais du Sang</italic>, EFS). None of the controls had had an infection (bacterial, viral, fungal, yeast) or had been vaccinated in the 30 days preceding inclusion. The study was approved by the local Ethics Committee, and all participants gave written informed consent.</p>
</sec>
<sec id="s2_2">
<title>MP-enriched EV preparation</title>
<p>MP-enriched EVs were isolated as previously described (<xref ref-type="bibr" rid="B9">9</xref>, <xref ref-type="bibr" rid="B10">10</xref>, <xref ref-type="bibr" rid="B29">29</xref>), by differential centrifugation at an initial speed of 3,000 x <italic>g</italic> at 4&#xb0;C for 10 minutes. The supernatant was then centrifuged at 13,000 x <italic>g</italic> at 4&#xb0;C for 10 minutes for the preparation of a platelet-free supernatant. MP-enriched EVs were concentrated by centrifuging the platelet-free supernatant for 1 hour at 100,000 x <italic>g</italic> at 4&#xb0;C. MP-enriched EVs were resuspended in filtered PBS (filtration through a 0.1 &#x3bc;m pore-size PES membrane) and MPs were characterized by flow cytometry detection.</p>
</sec>
<sec id="s2_3">
<title>MP phenotyping</title>
<p>MPs were labeled as previously described (<xref ref-type="bibr" rid="B10">10</xref>, <xref ref-type="bibr" rid="B11">11</xref>, <xref ref-type="bibr" rid="B29">29</xref>), with fluorochrome-conjugated monoclonal antibodies. MPs were labeled with five different panels from the antibodies described in <xref ref-type="supplementary-material" rid="SM1"><bold>Supplemental Table S3</bold></xref>. Fluorescence was assessed with a 20-parameter LSR Fortessa flow cytometer with a small-particle option (BD Biosciences) based on photomultiplier (PMT)-coupled forward scatter (FSC) detection. This mode of detection was used to ensure the optimal detection of MPs with diameters of 200 to 900 nm. The performance of the flow cytometer was checked before each assay. Megamix-Plus FSC and SSC beads (BioCytex, Marseille, France) of known dimensions (with mean diameters of 200 nm, 500 nm and 900 nm) were used for the standardization of FSC-PMT parameters and definition of the MP gate. MPs were acquired at low speed and quantified in Trucount tubes (BD Biosciences).</p>
</sec>
<sec id="s2_4">
<title>Flow cytometry analysis</title>
<p>Flow cytometry data were analyzed with FlowJo software (v.10.7.1, Ashland, OR).</p>
</sec>
<sec id="s2_5">
<title>Statistical analysis</title>
<p>All analyses were performed with Prism 6.07 software (GraphPad Software, La Jolla, CA). Only significant differences between groups (<italic>P</italic>&lt;0.05) are indicated on the data plots.</p>
</sec>
</sec>
<sec id="s3" sec-type="results">
<title>Results</title>
<sec id="s3_1">
<title>Characterization of MPs in PLWH</title>
<p>We compared the profiles of MPs from fresh plasma between PLWH (<italic>n</italic> = 42) and controls (<italic>n</italic> = 21) (<xref ref-type="fig" rid="f1"><bold>Figure&#xa0;1</bold></xref>). The MP gate extended from just below the 200 nm bead signal up to the 900 nm bead signal on the side scatter (SSC) and photomultiplier (PMT)-forward scatter (FSC) dot plot (<xref ref-type="fig" rid="f1"><bold>Figure&#xa0;1A</bold></xref>). The number and size distribution of MPs were determined by considering three size classes: small (&lt; 300 nm), medium (300-500 nm), and large (&gt; 500 nm) MPs (<xref ref-type="fig" rid="f1"><bold>Figures&#xa0;1A&#x2013;C</bold></xref>).</p>
<fig id="f1" position="float">
<label>Figure&#xa0;1</label>
<caption>
<p>Distribution of MPs by size and cellular origin: comparison of PLWH and controls. <bold>(A)</bold> Example of the gating strategy used for MP phenotyping by flow cytometry for the control group (<italic>n</italic> = 21) and PLWH (<italic>n</italic> = 42) (21 experiments with 2 patients and 1 control per experiment). On the left, a dot plot showing the settings based on fluorescent beads for the differentiation of three particle sizes: 200, 500 and 900 nm in diameter. On the right, dot plot for MP acquisition on a Fortessa flow cytometer for one representative PLWH. <bold>(B)</bold> Total MPs and <bold>(C)</bold> MPs of various sizes were quantified by flow cytometry and their concentrations were determined with Trucount tubes. <bold>(D)</bold> Example of the gating strategy used for the cellular origin phenotyping of MPs by flow cytometry for the control group (<italic>n</italic> = 21) and PLWH (<italic>n</italic> = 42) (21 experiments with 2 patients and 1 control per experiment). RBC MPs, MMPs, PMPs, Neutro MPs, DC MPs, TL MPs, BL MPs and Endo MPs were acquired in the MP gate. The indicated percentages represent the cell subpopulations of the MPs out of the total population. Representative FACS plots for each cellular subtype of MPs are shown. <bold>(E)</bold> Each cellular subtype of MPs was quantified by flow cytometry and MP concentrations were determined with Trucount tubes. The cellular markers are arranged in descending order of the number of MPs/mL expressing them in plasma. Horizontal bars indicate the median values. <bold>(F)</bold> The frequencies of CD235a<sup>+</sup> RBC MP, CD14<sup>+</sup> MMPs, CD41a<sup>+</sup> PMPs, CD10<sup>+</sup> Neutro MPs, CD11c<sup>+</sup>, CD123<sup>+</sup> DC MPs, CD3<sup>+</sup> TL MPs, CD19<sup>+</sup> BL MPs and CD142<sup>+</sup> Endo MPs were determined by flow cytometry for the control group (<italic>n</italic> = 21) and PLWH (<italic>n</italic> = 42) (21 experiments, with 2 patients and 1 control per experiment). The percentages were reported on the totality of MPs of cellular origin studied. <italic>P</italic> values (<italic>P</italic>&lt;0.05 considered significant) were obtained in Mann-Whitney and <italic>post hoc</italic> tests. **** <italic>P</italic>&lt;0.0001, *** <italic>P</italic>&lt;0.001, ** P&lt;0.01, * <italic>P</italic>&lt;0.05.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fimmu-15-1354065-g001.tif"/>
</fig>
<p>PLWH had significantly more circulating MPs than the controls (13.1x10<sup>6</sup> &#xb1; 2.1x10<sup>6</sup> vs. 4.7x10<sup>6</sup> &#xb1; 0.7x10<sup>6</sup> MPs/ml plasma, <italic>P</italic>&lt;0.0001) (<xref ref-type="fig" rid="f1"><bold>Figure&#xa0;1B</bold></xref>). This large increase in the number of MPs concerned MPs of all sizes: small MPs, 8.8x10<sup>6</sup> &#xb1; 1.6x10<sup>6</sup> vs. 3.2x10<sup>6</sup> &#xb1; 0.6x10<sup>6</sup> MPs/ml plasma (<italic>P</italic>=0.0003); medium-sized MPs, 3.2x10<sup>6</sup> &#xb1; 0.5x10<sup>6</sup> vs. 1.0x10<sup>6</sup> &#xb1; 0.1x10<sup>6</sup> MPs/ml plasma (<italic>P</italic>&lt;0.0001); large MPs, 1.1x10<sup>6</sup> &#xb1; 0.1x10<sup>6</sup> vs. 0.5x10<sup>6</sup> &#xb1; 0.1x10<sup>6</sup> MPs/ml plasma (<italic>P</italic>&lt;0.0001) (<xref ref-type="fig" rid="f1"><bold>Figure&#xa0;1C</bold></xref>).</p>
</sec>
<sec id="s3_2">
<title>Cellular origin of MPs in PLWH</title>
<p>We analyzed subpopulations of MPs defined on the basis of their cellular origin, as determined by evaluating the expression of surface proteins specific to red blood cells (CD235a<sup>+</sup> RBC MPs), platelets (CD41a<sup>+</sup> PMPs), monocytes (CD14<sup>+</sup> MMPs), neutrophils (CD10<sup>+</sup> Neutro MPs), dendritic cells (CD11c<sup>+</sup>, CD123<sup>+</sup> DC MPs), T lymphocytes (CD3<sup>+</sup> TL MPs), B lymphocytes (CD19<sup>+</sup> BL MPs) and endothelial cells (CD142<sup>+</sup> Endo MPs) (<xref ref-type="fig" rid="f1"><bold>Figures&#xa0;1D&#x2013;F</bold></xref>).</p>
<p>The concentrations of MPs of all cell origins other than platelets or monocytes (PMPs or MMPs) were significantly higher in PLWH than in controls: for RBC MPs, 1.3x10<sup>6</sup> &#xb1; 0.1x10<sup>6</sup> vs. 0.4x10<sup>6</sup> &#xb1; 0.1x10<sup>6</sup> MPs/ml (<italic>P</italic>&lt;0.0001); for DC MPs, 14.5x10<sup>4</sup> &#xb1; 1.9x10<sup>4</sup> vs. 4.7x10<sup>4</sup> &#xb1; 0.8x10<sup>4</sup> MPs/ml (<italic>P</italic>&lt;0.0001); for Neutro MPs, 20.6x10<sup>4</sup> &#xb1; 4.9x10<sup>4</sup> vs. 5.8x10<sup>4</sup> &#xb1; 1.6x10<sup>4</sup> MPs/ml (<italic>P</italic>=0.0101); for TL MPs, 13.6x10<sup>4</sup> &#xb1; 1.7x10<sup>4</sup> vs. 3.6x10<sup>4</sup> &#xb1; 0.5x10<sup>4</sup> MPs/ml (<italic>P</italic>&lt;0.0001); for BL MPs, 5.8x10<sup>4</sup> &#xb1; 0.9x10<sup>4</sup> vs. 2.2x10<sup>4</sup> &#xb1; 0.5x10<sup>4</sup> MPs/ml (<italic>P</italic>=0.0007); for Endo MPs, 4.7x10<sup>4</sup> &#xb1; 0.7x10<sup>4</sup> vs. 1.8x10<sup>4</sup> &#xb1; 0.3x10<sup>4</sup> MPs/ml (<italic>P</italic>=0.0008) (<xref ref-type="fig" rid="f1"><bold>Figure&#xa0;1E</bold></xref>).</p>
<p>We also compared the frequencies of each subtype of MPs based on cell origin between PLWH and controls (<xref ref-type="fig" rid="f1"><bold>Figure&#xa0;1F</bold></xref>). RBC MPs, PMPs and MMPs were each present at frequencies of more than 10% and together accounted for about 80% of the MPs studied in blood (<xref ref-type="fig" rid="f1"><bold>Figure&#xa0;1F</bold></xref>). The frequencies of RBC MPs and TL MPs were significantly higher in PLWH than in controls (RBC MPs, 53.2% &#xb1; 2.4% vs. 40.7% &#xb1; 3.1%, <italic>P</italic>=0.004); TL MPs, 5.9% &#xb1; 0.6% vs. 3.8% &#xb1; 0.5%, <italic>P</italic>=0.034). However, the frequencies of MMPs and PMPs were significantly lower in PLWH than in controls (MMPs, 12.8% &#xb1; 1.4% vs. 22.8% &#xb1; 3.4%, <italic>P</italic>=0.008; PMPs, 10.6% &#xb1; 1.0% vs. 17.7% &#xb1; 2.3%, <italic>P</italic>=0.006) (<xref ref-type="fig" rid="f1"><bold>Figure&#xa0;1F</bold></xref>). We found no correlation between cellular and MP phenotypes, either in PLWH or in the control group (data not shown).</p>
</sec>
<sec id="s3_3">
<title>Immunoregulatory profile of total MPs in PLWH</title>
<p>We characterized the immunoregulatory profile of total MPs from PLWH and controls. We performed flow cytometry to assess the membrane expression of 23 cellular activation and immune checkpoint markers potentially involved in chronic immune activation (PD1, ICOSL, OX40L, CD40L, LAG3, TGF-&#x3b2;1, PDL1, PDL2, HLA-ABC, CD86, DC-SIGN, CLEC2, CXCR4, CCR5, CD40, OX40, FASL, TIM3, HLA-DR, TRAIL, CTLA4, CD27 and CD39) (<xref ref-type="supplementary-material" rid="SM1"><bold>Supplementary Figures&#xa0;1</bold></xref>, <xref ref-type="supplementary-material" rid="SM1"><bold>2</bold></xref>, <xref ref-type="fig" rid="f2"><bold>Figure&#xa0;2</bold></xref>).</p>
<fig id="f2" position="float">
<label>Figure&#xa0;2</label>
<caption>
<p>Immunoregulatory molecule phenotypes of MPs from PLWH and the control group. The expression of immunoregulatory markers on the surface of total MPs <bold>(A)</bold>, CD41a<sup>+</sup> PMPs <bold>(B)</bold>, CD14<sup>+</sup> MMPs <bold>(C)</bold> and CD3<sup>+</sup> LMPs <bold>(D)</bold> was quantified by flow cytometry on fresh plasma samples from the control group (<italic>n</italic> = 21) and PLWH (<italic>n</italic> = 42) (21 experiments, with 2 patients and 1 control per experiment). The concentrations of MPs expressing each of the markers were determined with Trucount tubes. The immunoregulatory markers are arranged in descending order of the number of MPs/mL of plasma expressing them. Horizontal bars indicate the median values. <italic>P</italic> values (<italic>P</italic>&lt;0.05 considered significant) were obtained in Mann-Whitney and <italic>post hoc</italic> tests. **** <italic>P</italic>&lt;0.0001, *** <italic>P</italic>&lt;0.001, ** <italic>P</italic>&lt;0.01, * <italic>P</italic>&lt;0.05.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fimmu-15-1354065-g002.tif"/>
</fig>
<p>We identified 12 immunoregulatory molecules among the markers tested that were more strongly expressed by the MPs of PLWH than by those of controls (<xref ref-type="fig" rid="f2"><bold>Figure&#xa0;2A</bold></xref>). The markers on total MPs that did not differ significantly between groups are presented in <xref ref-type="supplementary-material" rid="SM1"><bold>Supplementary Figure&#xa0;2</bold></xref>.</p>
<p>For ligands of PD1, the differences between PLWH and the control group were as follows: for PDL1<sup>+</sup> MPs, 1.7x10<sup>5</sup> &#xb1; 0.3x10<sup>5</sup> vs. 0.5x10<sup>5</sup> &#xb1; 0.1x10<sup>5</sup> MPs/ml, (<italic>P</italic>=0.0006), and for PDL2<sup>+</sup> MPs, 1.6x10<sup>4</sup> &#xb1; 0.3x10<sup>4</sup> vs. 0.7x10<sup>4</sup> &#xb1; 0.1x10<sup>4</sup> MPs/ml (<italic>P</italic>=0.003).</p>
<p>For MHC I and II molecules, the differences in expression on MPs between PLWH and controls were as follows: for HLA-ABC<sup>+</sup> MPs, 2.2x10<sup>5</sup> &#xb1; 0.3x10<sup>5</sup> vs. 0.8x10<sup>5</sup> &#xb1; 0.3x10<sup>5</sup> MPs/ml (<italic>P</italic>&lt;0.0001), and for HLA-DR<sup>+</sup> MPs, 1.3x10<sup>5</sup> &#xb1; 0.2x10<sup>5</sup> vs. 0.4x10<sup>5</sup> &#xb1; 0.1x10<sup>5</sup> MPs/ml (<italic>P</italic>&lt;0.0001).</p>
<p>For the costimulation molecules ICOSL and OX40, the differences between PLWH and controls were as follows: for ICOSL<sup>+</sup> MPs, 2.2x10<sup>5</sup> &#xb1; 0.9x10<sup>5</sup> vs. 0.7x10<sup>5</sup> &#xb1; 0.2x10<sup>5</sup> MPs/ml (<italic>P</italic>=0.016), and for OX40<sup>+</sup> MPs, 5.5x10<sup>4</sup> &#xb1; 0.1x10<sup>4</sup> vs. 2.4x10<sup>4</sup> &#xb1; 0.1x10<sup>4</sup> MPs/ml (<italic>P</italic>=0.008).</p>
<p>For the other costimulation molecules considered, TGF-&#x3b2;1, TRAIL and CTLA4, the differences between PLWH controls were as follows: for TGF-&#x3b2;1<sup>+</sup> MPs, 1.6x10<sup>5</sup> &#xb1; 0.3x10<sup>5</sup> vs. 0.4x10<sup>5</sup> &#xb1; 0.1x10<sup>5</sup> MPs/ml (<italic>P</italic>=0.002); for TRAIL<sup>+</sup> MPs, 1.3x10<sup>5</sup> &#xb1; 0.3x10<sup>5</sup> vs. 0.5x10<sup>5</sup> &#xb1; 0.1x10<sup>5</sup> MPs/ml (<italic>P</italic>=0.0241), and for CTLA4<sup>+</sup> MPs, 2.3x10<sup>4</sup> &#xb1; 0.3x10<sup>4</sup> vs. 1.0x10<sup>4</sup> &#xb1; 0.1x10<sup>4</sup> MPs/ml (<italic>P</italic>=0.002).</p>
<p>Chemokine receptors were also more strongly expressed on the MPs of PLWH than on those of controls: for CCR5<sup>+</sup> MPs, 2.0x10<sup>5</sup> &#xb1; 0.3x10<sup>5</sup> vs. 0.9x10<sup>5</sup> &#xb1; 0.2x10<sup>5</sup> MPs/ml plasma (<italic>P</italic>=0.0003), and for CXCR4<sup>+</sup> MPs, 6.3x10<sup>4</sup> &#xb1; 0.1x10<sup>4</sup> vs. 2.8x10<sup>4</sup> &#xb1; 0.5x10<sup>4</sup> MPs/ml plasma (<italic>P</italic>=0.039).</p>
<p>DC-SIGN was also more strongly expressed on the MPs from PLWH than on those of controls: 3.4x10<sup>4</sup> &#xb1; 0.8x10<sup>4</sup> vs. 1.9x10<sup>4</sup> &#xb1; 0.3x10<sup>4</sup> MPs/ml plasma (<italic>P</italic>=0.04).</p>
</sec>
<sec id="s3_4">
<title>Immunoregulatory profiles of PMPs, MMPs and LMPs in PLWH</title>
<p>We then evaluated the immunoregulatory profile of MPs according to their cell type of origin, focusing particularly on PMPs, MMPs and LMPs, which are derived from cell subsets known to be involved in immunoregulation (<xref ref-type="bibr" rid="B8">8</xref>&#x2013;<xref ref-type="bibr" rid="B11">11</xref>, <xref ref-type="bibr" rid="B13">13</xref>, <xref ref-type="bibr" rid="B14">14</xref>).</p>
<sec id="s3_4_1">
<title>CD41a<sup>+</sup> PMPs</title>
<p>MPs from platelets can account for up to 32% of the MPs circulating in the blood of PLWH (<xref ref-type="fig" rid="f1"><bold>Figure&#xa0;1F</bold></xref>) and are therefore a potentially important factor in innate and adaptive immunity. The immunoregulatory molecule expression profile of the PMPs of PLWH was characterized by flow cytometry based on 15 surface markers: HLA-ABC, TGF&#x3b2;1, CXCR4, ICOSL, CLEC2, DC-SIGN, CD40, CD86, OX40, OX40L, CCR5, CD39, CD40L, LAG3, PD1 (<xref ref-type="supplementary-material" rid="SM1"><bold>Supplementary Figure&#xa0;3</bold></xref>, <xref ref-type="fig" rid="f2"><bold>Figure&#xa0;2B</bold></xref>). We observed a significant overexpression of five immunoregulatory molecules on the PMPs of PLWH relative to controls: HLA-ABC, 8.6x10<sup>4</sup> &#xb1; 1.6x10<sup>4</sup> vs. 4.8x10<sup>4</sup> &#xb1; 2.0x10<sup>4</sup> MPs/ml (<italic>P</italic>=0.004); TGF-&#x3b2;1, 3.6x10<sup>4</sup> &#xb1; 1.5x10<sup>4</sup> vs. 0.9x10<sup>4</sup> &#xb1; 0.2x10<sup>4</sup> MPs/ml (<italic>P</italic>=0.014); OX40, 4.7x10<sup>3</sup> &#xb1; 0.9x10<sup>3</sup> vs. 3.0x10<sup>3</sup> &#xb1; 1.3x10<sup>3</sup> MPs/ml (<italic>P</italic>=0.04); CD40L, 1.4x10<sup>3</sup> &#xb1; 0.3x10<sup>3</sup> vs. 0.7x10<sup>3</sup> &#xb1; 0.1x10<sup>3</sup> MPs/ml (<italic>P</italic>=0.03) and LAG3, 7.4x10<sup>3</sup> &#xb1; 4.8x10<sup>3</sup> vs. 0.7x10<sup>3</sup> &#xb1; 0.1x10<sup>3</sup> MPs/ml (<italic>P</italic>=0.002) (<xref ref-type="fig" rid="f2"><bold>Figure&#xa0;2B</bold></xref>). The markers on PMPs that did not differ significantly between groups are presented in <xref ref-type="supplementary-material" rid="SM1"><bold>Supplementary Figure&#xa0;3</bold></xref>.</p>
</sec>
<sec id="s3_4_2">
<title>CD14<sup>+</sup> MMPs</title>
<p>MPs derived from monocytes can account for up to 38% of the circulating MPs in the blood of PLWH (<xref ref-type="fig" rid="f1"><bold>Figure&#xa0;1F</bold></xref>) and may have effects on inflammation, oxidative stress and cell apoptosis. We performed flow cytometry to characterize the immunoregulatory molecule profiles of MMPs for 16 markers: PDL1, HLA-DR, TRAIL, OX40L, ICOSL, FASL, CD86, CCR5, TIM3, CXCR4, CD39, PDL2, TGF&#x3b2;1, LAG3, CD40L, PD1 (<xref ref-type="supplementary-material" rid="SM1"><bold>Supplementary Figure&#xa0;4</bold></xref>, <xref ref-type="fig" rid="f2"><bold>Figure&#xa0;2C</bold></xref>). Five immunoregulatory molecules were significantly overexpressed on MMPs from PLWH relative to MMPs from the control group: HLA-DR, 2.5x10<sup>4</sup> &#xb1; 0.7x10<sup>4</sup> vs. 0.5x10<sup>4</sup> &#xb1; 0.1x10<sup>4</sup> MPs/ml (<italic>P</italic>=0.001); TRAIL, 2.5x10<sup>4</sup> &#xb1; 0.8x10<sup>4</sup> vs. 1.5x10<sup>4</sup> &#xb1; 0.7x10<sup>4</sup> MPs/ml (<italic>P</italic>=0.022); CCR5, 1.8x10<sup>4</sup> &#xb1; 0.4x10<sup>4</sup> vs. 0.4x10<sup>4</sup> &#xb1; 0.1x10<sup>4</sup> MPs/ml (<italic>P</italic>=0.001); TIM3, 1.4x10<sup>4</sup> &#xb1; 0.8x10<sup>4</sup> vs. 0.4x10<sup>4</sup> &#xb1; 0.1x10<sup>4</sup> MPs/ml (<italic>P</italic>=0.044), and PDL2, 5.8x10<sup>3</sup> &#xb1; 0.8x10<sup>3</sup> vs. 2.5x10<sup>3</sup> &#xb1; 0.6x10<sup>3</sup> MPs/ml (<italic>P</italic>=0.005) (<xref ref-type="fig" rid="f2"><bold>Figure&#xa0;2C</bold></xref>). The markers on MMPs that did not differ significantly between groups are presented in <xref ref-type="supplementary-material" rid="SM1"><bold>Supplementary Figure&#xa0;4</bold></xref>.</p>
</sec>
<sec id="s3_4_3">
<title>CD3<sup>+</sup> LMPs</title>
<p>MPs derived from lymphocytes can account for up to 18% of the MPs circulating in the blood of PLWH (<xref ref-type="fig" rid="f1"><bold>Figure&#xa0;1F</bold></xref>). The mean frequency of LMPs among total circulating MPs is only 6%, but even small numbers of LMPs can exert immunomodulatory effects, particularly on lymphocytes. The surface immunoregulatory molecule profile of the LMPs was characterized by flow cytometry with 12 markers: OX40L, CD39, ICOSL, FASL, PDL1, CD27, LAG3, TGF&#x3b2;1, TRAIL, CD40L, PD1 and CTLA-4 (<xref ref-type="supplementary-material" rid="SM1"><bold>Supplementary Figure&#xa0;5</bold></xref>, <xref ref-type="fig" rid="f2"><bold>Figure&#xa0;2D</bold></xref>). We found that six immunomodulatory molecules were more strongly expressed on LMPs from PLWH than on LMPs from controls: OX40L, 1.6x10<sup>4</sup> &#xb1; 0.3x10<sup>4</sup> vs. 0.7x10<sup>4</sup> &#xb1; 0.2x10<sup>4</sup> MPs/ml (<italic>P</italic>=0.01); ICOSL, 6.2x10<sup>3</sup> &#xb1; 1.3x10<sup>3</sup> vs. 2.9x10<sup>3</sup> &#xb1; 0.7x10<sup>3</sup> MPs/ml (<italic>P</italic>=0.01); FASL, 11.3x10<sup>3</sup> &#xb1; 1.9x10<sup>3</sup> vs. 3.0x10<sup>3</sup> &#xb1; 0.6x10<sup>3</sup> MPs/ml (<italic>P</italic>=0.0002); PDL1, 8.8x10<sup>3</sup> &#xb1; 1.6x10<sup>3</sup> vs. 2.5x10<sup>3</sup> &#xb1; 0.7x10<sup>3</sup> MPs/ml (<italic>P</italic>=0.0009); CD27, 3.9x10<sup>3</sup> &#xb1; 0.6x10<sup>3</sup> vs. 1.5x10<sup>3</sup> &#xb1; 0.3x10<sup>3</sup> MPs/ml (<italic>P</italic>=0.001), and CTLA4, 14.7x10<sup>2</sup> &#xb1; 2.6x10<sup>2</sup> vs. 4.0x10<sup>2</sup> &#xb1; 1.1x10<sup>2</sup> MPs/ml (<italic>P</italic>=0.001). (<xref ref-type="fig" rid="f2"><bold>Figure&#xa0;2D</bold></xref>). The markers on LMPs that did not differ significantly between groups are presented in <xref ref-type="supplementary-material" rid="SM1"><bold>Supplementary Figure&#xa0;5</bold></xref>.</p>
<p>Finally, despite the identical activation profiles of PLWH and control MPs (<xref ref-type="supplementary-material" rid="SM1"><bold>Supplementary Figure&#xa0;6</bold></xref>), the number of immunomodulatory MPs was greater in PLWH than controls.</p>
</sec>
</sec>
</sec>
<sec id="s4" sec-type="discussion">
<title>Discussion</title>
<p>We found that PLWH on ART had significantly larger numbers of MPs in their plasma than controls. This increase in MP numbers, which points to cellular activation, is potentially important because MPs can have various effects, depending on their phenotype and numbers (<xref ref-type="bibr" rid="B9">9</xref>&#x2013;<xref ref-type="bibr" rid="B11">11</xref>). Many factors, including aging, can influence the production of MPs even in people without HIV (<xref ref-type="bibr" rid="B30">30</xref>). However, we observed no correlation between age and the number of MPs in either PLWH or controls (data not shown).</p>
<p>We found that the increase in the number of MPs in PLWH was not dependent on MP size, with all size classes, including the largest (900 nm), affected. This finding is important, because the largest MPs may contain organelles, such as mitochondria, which may participate in the production of anti-cardiolipin antibodies (<xref ref-type="bibr" rid="B12">12</xref>). These antibodies play an important role in anti-phospholipid antibody syndrome and are responsible for the development of a state of thrombophilia. Other MPs may contain proteasomes, which can contribute to antigen presentation through class I MHC (<xref ref-type="bibr" rid="B14">14</xref>).</p>
<p>Moreover, the phenotypic characteristics of MPs are key to identification of the cellular targets of MPs in the immune system. Indeed, the interactions of MPs with immune cells differ according to the cellular origin of the MPs and their immunoregulatory phenotype (<xref ref-type="bibr" rid="B9">9</xref>, <xref ref-type="bibr" rid="B11">11</xref>). The interaction between MPs and immune cells is not random and involves molecules present on the surface of MPs (<xref ref-type="bibr" rid="B11">11</xref>). This interaction can result in a transfer of molecules from the MP to the immune cell, with immunomodulatory consequences. We show here that the increase in MP numbers in PLWH concerns not only MPs from red blood cells and endothelial cells, but also other MPs derived from immune system cells, such as neutrophils, dendritic cells and T and B lymphocytes. This increase cannot be explained by an increase in the number of cells because we have never observed any correlation between cellular expression and MPs, in either controls (<xref ref-type="bibr" rid="B9">9</xref>) or PLWH (data not shown). Furthermore, we have recently shown that the role of MPs depends on their quantity (<xref ref-type="bibr" rid="B9">9</xref>). We have shown that LMPs, even if present at only very low levels, can facilitate both cellular and humoral responses. These findings support the possible maintenance of an inflammatory endothelial environment, and immune activation through specific interactions of MPs with the immune system (<xref ref-type="bibr" rid="B11">11</xref>).</p>
<p>Only MPs derived from monocytes and platelets were present in similar numbers in both the PLWH and control groups. However, the phenotype of these MPs differed between PLWH and controls. Indeed, the analysis of immunomodulatory molecule expression on the surface of PMPs indicated that class I MHC molecules (HLA-ABC), TGF&#x3b2;1, OX40 (CD134), LAG3 (CD223) and CD40L (CD154) were more strongly expressed on the PMPs of PLWH than on those of controls. These data confirm the state of chronic platelet activation described in PLWH patients (<xref ref-type="bibr" rid="B31">31</xref>) and also clearly suggest a role for these MPs in immune activation (<xref ref-type="bibr" rid="B9">9</xref>).</p>
<p>Differences in the immunomodulatory molecules expressed on the surface were also found for MMPs, but for the molecules concerned were different, with an overexpression of molecules capable of modulating immune activation, such as class II MHC (HLA-DR), TRAIL (CD253), TIM3 (CD366) and PDL2 (CD273) in PLWH relative to controls. The MMPs of PLWH also had higher levels of CCR5 expression on their surface. MMPs could, thus, play a key role in the spread of the virus and the maintenance of virus reservoirs (<xref ref-type="bibr" rid="B14">14</xref>, <xref ref-type="bibr" rid="B18">18</xref>).</p>
<p>Similarly, LMPs from PLWH had significantly higher levels of expression for OX40L (CD252), ICOSL (CD275), FASL (CD178), PDL1 (CD274), CTLA4 (CD152) and CD27 than those from controls. These findings clearly suggest that these MPs could play a role in the transfer or maintenance of T-lymphocyte immune activation, as reported in our previous studies (<xref ref-type="bibr" rid="B11">11</xref>). Indeed, CD27<sup>+</sup> MPs bind only to cells that already express CD27. This binding results partly from the co-expression of CD70 by these CD27<sup>+</sup> MPs (<xref ref-type="bibr" rid="B11">11</xref>). These data testify to the ability of MPs to reprogram cells via marker transfer, thereby maintaining an immune phenotype.</p>
<p>We also observed much stronger DC-SIGN and CXCR4 expression on MPs from PLWH than on those from controls. The cellular origin of the MPs overexpressing these two markers remains unclear, but we suspect that they are derived from dendritic cells and/or B lymphocytes and can participate in the maintenance and dissemination of the virus. Furthermore, it has been known since the early 2000s that CXCR4 and CCR5 are present on MPs and can transfer a capacity for infection to cells lacking these receptors (<xref ref-type="bibr" rid="B18">18</xref>, <xref ref-type="bibr" rid="B19">19</xref>). This ability of MPs to transfer markers suggests that DC-SIGN and CXCR4 MPs may be associated with the maintenance of a viral load in reservoirs.</p>
<p>Despite the careful application of selection criteria during the recruitment of participants (HIV infection, patient on treatment with subsequent immune reconstitution), phenotyping results for MPs were highly heterogeneous, particularly for PDL1, TGF&#x3b2;1 and CTLA4. This variability suggests that comorbid conditions in these patients may drive the characteristic expression of immunomodulatory markers on the surface of MPs. Indeed, these MPs with particular phenotypes may be associated with comorbid conditions and chronic activation of the immune system.</p>
<p>Other chronic co-infections and the resulting activation of the immune system may also explain these higher levels of MPs, but we found no link between the number of MPs and chronic co-infection with HBV or HCV (data not shown).</p>
<p>Finally, we hypothesize that antiviral treatments may direct the production of MPs. We found no correlation between the frequency or phenotype of MPs and ART treatment (data not shown), but we believe that this lack of correlation may result from treatment heterogeneity, which might also account for the occurrence of comorbidities as a function of the MPs induced. The only information available to support this hypothesis comes from studies on the effects on RBCs of ART. Indeed, Peltenburg et&#xa0;al. have shown that abacavir treatment is associated with an increase in cardiovascular risk, whereas no such increase in risk is observed for tenofovir or didanosine (<xref ref-type="bibr" rid="B32">32</xref>, <xref ref-type="bibr" rid="B33">33</xref>). However, it remains unclear whether this risk is associated with RBC-MPs. There are also indirect links, such as the impact on cholesterol and, hence, on MP survival (<xref ref-type="bibr" rid="B34">34</xref>), and the effect of nucleoside reverse transcriptase inhibitors on RBC (<xref ref-type="bibr" rid="B35">35</xref>). Dolutegravir is also directly associated with the apoptosis of RBCs (<xref ref-type="bibr" rid="B36">36</xref>) or other cell types (<xref ref-type="bibr" rid="B37">37</xref>), inflammation (<xref ref-type="bibr" rid="B38">38</xref>), and platelet activation correlated with an increase in the risk of cardiometabolic comorbidities (<xref ref-type="bibr" rid="B39">39</xref>). This last point links platelet activation and the production of PMPs (<xref ref-type="bibr" rid="B12">12</xref>).</p>
<p>The data obtained here are therefore of potential value for the development of new treatments for reducing the impact of these MPs, whether or not they are linked to infection. Indeed, new therapeutic or vaccination strategies based on the specific transfer of functions from MPs could be envisaged (<xref ref-type="bibr" rid="B11">11</xref>). However, as we recently showed with CD27<sup>+</sup> MPs, it is important to take the contribution of immunoregulatory MPs into account, particularly in patients treated with immunotherapies based on monoclonal antibodies (<xref ref-type="bibr" rid="B11">11</xref>).</p>
</sec>
<sec id="s5" sec-type="data-availability">
<title>Data availability statement</title>
<p>The original contributions presented in the study are included in the article/<xref ref-type="supplementary-material" rid="SM1"><bold>Supplementary Material</bold></xref>. Further inquiries can be directed to the corresponding author.</p>
</sec>
<sec id="s6" sec-type="ethics-statement">
<title>Ethics statement</title>
<p>The studies involving humans were approved by Comite de Protection des Personnes - Ile-de-France IX. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.</p>
</sec>
<sec id="s7" sec-type="author-contributions">
<title>Author contributions</title>
<p>DN-L: Formal analysis, Investigation, Writing &#x2013; original draft. MT: Writing &#x2013; review &amp; editing. RB: Writing &#x2013; review &amp; editing. SM: Writing &#x2013; review &amp; editing, Investigation. PV: Writing &#x2013; review &amp; editing, Investigation. JL: Writing &#x2013; review &amp; editing, Investigation. AD: Writing &#x2013; review &amp; editing. MA: Writing &#x2013; review &amp; editing. EB: Writing &#x2013; review &amp; editing. FC: Writing &#x2013; review &amp; editing. HH-C: Writing &#x2013; review &amp; editing. SP: Writing &#x2013; review &amp; editing. J-DL: Writing &#x2013; review &amp; editing. FP: Writing &#x2013; review &amp; editing. SG: Writing &#x2013; review &amp; editing. BV: Conceptualization, Supervision, Validation, Writing &#x2013; original draft.</p>
</sec>
</body>
<back>
<sec id="s8" sec-type="funding-information">
<title>Funding</title>
<p>The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was supported by the ANRS, <italic>Etablissement Franc&#x327;ais du Sang, INSERM and Universite&#x301; Paris-Est Cre&#x301;teil</italic>.</p>
</sec>
<ack>
<title>Acknowledgments</title>
<p>We are particularly grateful to the healthy blood donors who participated in this study, and the EFS team responsible for collecting blood donations.</p>
</ack>
<sec id="s9" sec-type="COI-statement">
<title>Conflict of interest</title>
<p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p>
</sec>
<sec id="s10" sec-type="disclaimer">
<title>Publisher&#x2019;s note</title>
<p>All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.</p>
</sec>
<sec id="s11" sec-type="supplementary-material">
<title>Supplementary material</title>
<p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type="uri" xlink:href="https://www.frontiersin.org/articles/10.3389/fimmu.2024.1354065/full#supplementary-material">https://www.frontiersin.org/articles/10.3389/fimmu.2024.1354065/full#supplementary-material</ext-link>
</p>
<supplementary-material xlink:href="DataSheet_1.docx" id="SM1" mimetype="application/vnd.openxmlformats-officedocument.wordprocessingml.document"/>
</sec>
<fn-group>
<title>Abbreviations</title>
<fn fn-type="abbr">
<p>PLWH, people living with HIV; ART, antiretroviral treatment; EV, extracellular vesicles; MPs, microvesicles; PMPs, platelet microvesicles; MMPs, monocyte microvesicles; LMPs, lymphocyte microvesicles; TL, T lymphocyte; BL, B lymphocyte; RBC, red blood cell; Neutro, neutrophil; DCs, dendritic cells; Endo, endothelial cells.</p>
</fn>
</fn-group>
<ref-list>
<title>References</title>
<ref id="B1">
<label>1</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Appay</surname> <given-names>V</given-names>
</name>
<name>
<surname>Kelleher</surname> <given-names>A</given-names>
</name>
</person-group>. <article-title>Immune activation and immune aging in HIV infection</article-title>. <source>Curr Opin HIV AIDS</source>. (<year>2016</year>) <volume>11</volume>:<page-range>242&#x2013;9</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1097/COH.0000000000000240</pub-id>
</citation>
</ref>
<ref id="B2">
<label>2</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Dias</surname> <given-names>M</given-names>
</name>
<name>
<surname>Costa</surname> <given-names>C</given-names>
</name>
<name>
<surname>daSilva</surname> <given-names>L</given-names>
</name>
</person-group>. <article-title>The ambiguous roles of extracellular vesicles in HIV replication and pathogenesis</article-title>. <source>Front Microbiol</source>. (<year>2018</year>) <volume>9</volume>:<elocation-id>2411</elocation-id>. doi:&#xa0;<pub-id pub-id-type="doi">10.3389/fmicb.2018.02411</pub-id>
</citation>
</ref>
<ref id="B3">
<label>3</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Colombo</surname> <given-names>M</given-names>
</name>
<name>
<surname>Raposo</surname> <given-names>G</given-names>
</name>
<name>
<surname>Th&#xe9;ry</surname> <given-names>C</given-names>
</name>
</person-group>. <article-title>Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles</article-title>. <source>Annu Rev Cell Dev Biol</source>. (<year>2014</year>) <volume>30</volume>:<page-range>255&#x2013;89</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1146/annurev-cellbio-101512-122326</pub-id>
</citation>
</ref>
<ref id="B4">
<label>4</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>van Niel</surname> <given-names>G</given-names>
</name>
<name>
<surname>D&#x2019;Angelo</surname> <given-names>G</given-names>
</name>
<name>
<surname>Raposo</surname> <given-names>G</given-names>
</name>
</person-group>. <article-title>Shedding light on the cell biology of extracellular vesicles</article-title>. <source>Nat Rev Mol Cell Biol</source>. (<year>2018</year>) <volume>19</volume>:<page-range>213&#x2013;28</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1038/nrm.2017.125</pub-id>
</citation>
</ref>
<ref id="B5">
<label>5</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Th&#xe9;ry</surname> <given-names>C</given-names>
</name>
<name>
<surname>Ostrowski</surname> <given-names>M</given-names>
</name>
<name>
<surname>Segura</surname> <given-names>E</given-names>
</name>
</person-group>. <article-title>Membrane vesicles as conveyors of immune responses</article-title>. <source>Nat Rev Immunol</source>. (<year>2009</year>) <volume>9</volume>:<page-range>581&#x2013;93</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1038/nri2567</pub-id>
</citation>
</ref>
<ref id="B6">
<label>6</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Dieud&#xe9;</surname> <given-names>M</given-names>
</name>
<name>
<surname>Bell</surname> <given-names>C</given-names>
</name>
<name>
<surname>Turgeon</surname> <given-names>J</given-names>
</name>
<name>
<surname>Beillevaire</surname> <given-names>D</given-names>
</name>
<name>
<surname>Pomerleau</surname> <given-names>L</given-names>
</name>
<name>
<surname>Yang</surname> <given-names>B</given-names>
</name>
<etal/>
</person-group>. <article-title>The 20S proteasome core, active within apoptotic exosome-like vesicles, induces autoantibody production and accelerates rejection</article-title>. <source>Sci Trans Med</source>. (<year>2015</year>) <volume>7</volume>:<fpage>200</fpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.1126/scitranslmed.aac9816</pub-id>
</citation>
</ref>
<ref id="B7">
<label>7</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Boudreau</surname> <given-names>L</given-names>
</name>
<name>
<surname>Duchez</surname> <given-names>A</given-names>
</name>
<name>
<surname>Cloutier</surname> <given-names>N</given-names>
</name>
<name>
<surname>Soulet</surname> <given-names>D</given-names>
</name>
<name>
<surname>Martin</surname> <given-names>N</given-names>
</name>
<name>
<surname>Bollinger</surname> <given-names>J</given-names>
</name>
<etal/>
</person-group>. <article-title>Platelets release mitochondria serving as substrate for bactericidal group IIA-secreted phospholipase A2 to promote inflammation</article-title>. <source>Blood</source>. (<year>2014</year>) <volume>124</volume>:<page-range>2173&#x2013;83</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1182/blood-2014-05-573543</pub-id>
</citation>
</ref>
<ref id="B8">
<label>8</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Danesh</surname> <given-names>A</given-names>
</name>
<name>
<surname>Inglis</surname> <given-names>H</given-names>
</name>
<name>
<surname>Jackman</surname> <given-names>R</given-names>
</name>
<name>
<surname>Wu</surname> <given-names>S</given-names>
</name>
<name>
<surname>Deng</surname> <given-names>X</given-names>
</name>
<name>
<surname>Muench</surname> <given-names>M</given-names>
</name>
<etal/>
</person-group>. <article-title>Exosomes from red blood cell units bind to monocytes and induce proinflammatory cytokines, boosting T-cell responses in vitro</article-title>. <source>Blood</source>. (<year>2014</year>) <volume>123</volume>:<page-range>687&#x2013;96</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1182/blood-2013-10-530469</pub-id>
</citation>
</ref>
<ref id="B9">
<label>9</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Pinheiro</surname> <given-names>M</given-names>
</name>
<name>
<surname>Tamagne</surname> <given-names>M</given-names>
</name>
<name>
<surname>Elayeb</surname> <given-names>R</given-names>
</name>
<name>
<surname>Andrieu</surname> <given-names>M</given-names>
</name>
<name>
<surname>Pirenne</surname> <given-names>F</given-names>
</name>
<name>
<surname>Vingert</surname> <given-names>B</given-names>
</name>
</person-group>. <article-title>Blood microparticles are a component of immune modulation in red blood cell transfusion</article-title>. <source>Eur J Immunol</source>. (<year>2020</year>) <volume>50</volume>:<page-range>1237&#x2013;40</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1002/eji.201948481</pub-id>
</citation>
</ref>
<ref id="B10">
<label>10</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Neyrinck-Leglantier</surname> <given-names>D</given-names>
</name>
<name>
<surname>Tamagne</surname> <given-names>M</given-names>
</name>
<name>
<surname>L&#x2019;honor&#xe9;</surname> <given-names>S</given-names>
</name>
<name>
<surname>Cagnet</surname> <given-names>L</given-names>
</name>
<name>
<surname>Pakdaman</surname> <given-names>S</given-names>
</name>
<name>
<surname>Marchand</surname> <given-names>A</given-names>
</name>
<etal/>
</person-group>. <article-title>Autologous blood extracellular vesicles and specific CD4+ T-cell co-activation</article-title>. <source>Front Immunol</source>. (<year>2022</year>) <volume>13</volume>:<elocation-id>992483</elocation-id>. doi:&#xa0;<pub-id pub-id-type="doi">10.3389/fimmu.2022.992483</pub-id>
</citation>
</ref>
<ref id="B11">
<label>11</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Cagnet</surname> <given-names>L</given-names>
</name>
<name>
<surname>Neyrinck-Leglantier</surname> <given-names>D</given-names>
</name>
<name>
<surname>Tamagne</surname> <given-names>M</given-names>
</name>
<name>
<surname>Berradhia</surname> <given-names>L</given-names>
</name>
<name>
<surname>Khelfa</surname> <given-names>M</given-names>
</name>
<name>
<surname>Cleophax</surname> <given-names>S</given-names>
</name>
<etal/>
</person-group>. <article-title>CD27+ microparticle interactions and immunoregulation of CD4+ T lymphocytes</article-title>. <source>Front Immunol</source>. (<year>2023</year>) <volume>14</volume>:<elocation-id>1043255</elocation-id>. doi:&#xa0;<pub-id pub-id-type="doi">10.3389/fimmu.2023.1043255</pub-id>
</citation>
</ref>
<ref id="B12">
<label>12</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Melki</surname> <given-names>I</given-names>
</name>
<name>
<surname>Tessandier</surname> <given-names>N</given-names>
</name>
<name>
<surname>Zufferey</surname> <given-names>A</given-names>
</name>
<name>
<surname>Boilard</surname> <given-names>E</given-names>
</name>
</person-group>. <article-title>Platelet microvesicles in health and disease</article-title>. <source>Platelets</source>. (<year>2017</year>) <volume>28</volume>:<page-range>214&#x2013;21</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1080/09537104.2016.1265924</pub-id>
</citation>
</ref>
<ref id="B13">
<label>13</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Dinkla</surname> <given-names>S</given-names>
</name>
<name>
<surname>van Cranenbroek</surname> <given-names>B</given-names>
</name>
<name>
<surname>van der Heijden</surname> <given-names>W</given-names>
</name>
<name>
<surname>He</surname> <given-names>X</given-names>
</name>
<name>
<surname>Wallbrecher</surname> <given-names>R</given-names>
</name>
<name>
<surname>Dumitriu</surname> <given-names>I</given-names>
</name>
<etal/>
</person-group>. <article-title>Platelet microparticles inhibit IL-17 production by regulatory T cells through P-selectin</article-title>. <source>Blood</source>. (<year>2016</year>) <volume>127</volume>:<page-range>1976&#x2013;86</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1182/blood-2015-04-640300</pub-id>
</citation>
</ref>
<ref id="B14">
<label>14</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Marcoux</surname> <given-names>G</given-names>
</name>
<name>
<surname>Laroche</surname> <given-names>A</given-names>
</name>
<name>
<surname>Hasse</surname> <given-names>S</given-names>
</name>
<name>
<surname>Bellio</surname> <given-names>M</given-names>
</name>
<name>
<surname>Mbarik</surname> <given-names>M</given-names>
</name>
<name>
<surname>Tamagne</surname> <given-names>M</given-names>
</name>
<etal/>
</person-group>. <article-title>Platelet EVs contain an active proteasome involved in protein processing for antigen presentation via MHC-I molecules</article-title>. <source>Blood</source>. (<year>2021</year>) <volume>138</volume>:<page-range>2607&#x2013;20</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1182/blood.2020009957</pub-id>
</citation>
</ref>
<ref id="B15">
<label>15</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Sadallah</surname> <given-names>S</given-names>
</name>
<name>
<surname>Eken</surname> <given-names>C</given-names>
</name>
<name>
<surname>Martin</surname> <given-names>P</given-names>
</name>
<name>
<surname>Schifferli</surname> <given-names>J</given-names>
</name>
</person-group>. <article-title>Microparticles (ectosomes) shed by stored human platelets downregulate macrophages and modify the development of dendritic cells</article-title>. <source>J Immunol</source>. (<year>2011</year>) <volume>186</volume>:<page-range>6543&#x2013;52</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.4049/jimmunol.1002788</pub-id>
</citation>
</ref>
<ref id="B16">
<label>16</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Caobi</surname> <given-names>A</given-names>
</name>
<name>
<surname>Nair</surname> <given-names>M</given-names>
</name>
<name>
<surname>Raymond</surname> <given-names>A</given-names>
</name>
</person-group>. <article-title>Extracellular vesicles in the pathogenesis of viral infections in humans</article-title>. <source>Viruses</source>. (<year>2020</year>) <volume>12</volume>:<fpage>1200</fpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.3390/v12101200</pub-id>
</citation>
</ref>
<ref id="B17">
<label>17</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Al Sharif</surname> <given-names>S</given-names>
</name>
<name>
<surname>Pinto</surname> <given-names>D</given-names>
</name>
<name>
<surname>Mensah</surname> <given-names>G</given-names>
</name>
<name>
<surname>Dehbandi</surname> <given-names>F</given-names>
</name>
<name>
<surname>Khatkar</surname> <given-names>P</given-names>
</name>
<name>
<surname>Kim</surname> <given-names>Y</given-names>
</name>
<etal/>
</person-group>. <article-title>Extracellular vesicles in HTLV-1 communication: the story of an invisible messenger</article-title>. <source>Viruses</source>. (<year>2020</year>) <volume>12</volume>:<fpage>1422</fpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.3390/v12121422</pub-id>
</citation>
</ref>
<ref id="B18">
<label>18</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Mack</surname> <given-names>M</given-names>
</name>
<name>
<surname>Kleinschmidt</surname> <given-names>A</given-names>
</name>
<name>
<surname>Br&#xfc;hl</surname> <given-names>H</given-names>
</name>
<name>
<surname>Klier</surname> <given-names>C</given-names>
</name>
<name>
<surname>Nelson</surname> <given-names>P</given-names>
</name>
<name>
<surname>Cihak</surname> <given-names>J</given-names>
</name>
<etal/>
</person-group>. <article-title>Transfer of the chemokine receptor CCR5 between cells by membrane-derived microparticles: a mechanism for cellular human immunodeficiency virus 1 infection</article-title>. <source>Nat Med</source>. (<year>2000</year>) <volume>6</volume>:<page-range>769&#x2013;75</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1038/77498</pub-id>
</citation>
</ref>
<ref id="B19">
<label>19</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Rozmyslowicz</surname> <given-names>T</given-names>
</name>
<name>
<surname>Majka</surname> <given-names>M</given-names>
</name>
<name>
<surname>Kijowski</surname> <given-names>J</given-names>
</name>
<name>
<surname>Murphy</surname> <given-names>S</given-names>
</name>
<name>
<surname>Conover</surname> <given-names>D</given-names>
</name>
<name>
<surname>Poncz</surname> <given-names>M</given-names>
</name>
<etal/>
</person-group>. <article-title>Platelet- and megakaryocyte-derived microparticles transfer CXCR4 receptor to CXCR4-null cells and make them susceptible to infection by X4-HIV</article-title>. <source>AIDS (London England)</source>. (<year>2003</year>) <volume>17</volume>:<fpage>33</fpage>&#x2013;<lpage>42</lpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.1097/00002030-200301030-00006</pub-id>
</citation>
</ref>
<ref id="B20">
<label>20</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Gasper-Smith</surname> <given-names>N</given-names>
</name>
<name>
<surname>Crossman</surname> <given-names>D</given-names>
</name>
<name>
<surname>Whitesides</surname> <given-names>J</given-names>
</name>
<name>
<surname>Mensali</surname> <given-names>N</given-names>
</name>
<name>
<surname>Ottinger</surname> <given-names>J</given-names>
</name>
<name>
<surname>Plonk</surname> <given-names>S</given-names>
</name>
<etal/>
</person-group>. <article-title>Induction of plasma (TRAIL), TNFR-2, Fas ligand, and plasma microparticles after human immunodeficiency virus type 1 (HIV-1) transmission: implications for HIV-1 vaccine design</article-title>. <source>J virology</source>. (<year>2008</year>) <volume>82</volume>:<page-range>7700&#x2013;10</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1128/JVI.00605-08</pub-id>
</citation>
</ref>
<ref id="B21">
<label>21</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Ruiz-de-Le&#xf3;n</surname> <given-names>M</given-names>
</name>
<name>
<surname>Jim&#xe9;nez-Sousa</surname> <given-names>M</given-names>
</name>
<name>
<surname>Moreno</surname> <given-names>S</given-names>
</name>
<name>
<surname>Garc&#xed;a</surname> <given-names>M</given-names>
</name>
<name>
<surname>Guti&#xe9;rrez-Rivas</surname> <given-names>M</given-names>
</name>
<name>
<surname>Le&#xf3;n</surname> <given-names>A</given-names>
</name>
<etal/>
</person-group>. <article-title>Lower expression of plasma-derived exosome miR-21 levels in HIV-1 elite controllers with decreasing CD4 T cell count</article-title>. <source>J microbiology immunology infection = Wei mian yu gan ran za zhi</source>. (<year>2019</year>) <volume>52</volume>:<page-range>667&#x2013;71</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1016/j.jmii.2018.07.007</pub-id>
</citation>
</ref>
<ref id="B22">
<label>22</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Chettimada</surname> <given-names>S</given-names>
</name>
<name>
<surname>Lorenz</surname> <given-names>D</given-names>
</name>
<name>
<surname>Misra</surname> <given-names>V</given-names>
</name>
<name>
<surname>Wolinsky</surname> <given-names>S</given-names>
</name>
<name>
<surname>Gabuzda</surname> <given-names>D</given-names>
</name>
</person-group>. <article-title>Small RNA sequencing of extracellular vesicles identifies circulating miRNAs related to inflammation and oxidative stress in HIV patients</article-title>. <source>BMC Immunol</source>. (<year>2020</year>) <volume>21</volume>:<fpage>57</fpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.1186/s12865-020-00386-5</pub-id>
</citation>
</ref>
<ref id="B23">
<label>23</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>DeMarino</surname> <given-names>C</given-names>
</name>
<name>
<surname>Pleet</surname> <given-names>M</given-names>
</name>
<name>
<surname>Cowen</surname> <given-names>M</given-names>
</name>
<name>
<surname>Barclay</surname> <given-names>R</given-names>
</name>
<name>
<surname>Akpamagbo</surname> <given-names>Y</given-names>
</name>
<name>
<surname>Erickson</surname> <given-names>J</given-names>
</name>
<etal/>
</person-group>. <article-title>Antiretroviral drugs alter the content of extracellular vesicles from HIV-1-infected cells</article-title>. <source>Sci Rep</source>. (<year>2018</year>) <volume>8</volume>:<fpage>7653</fpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.1038/s41598-018-25943-2</pub-id>
</citation>
</ref>
<ref id="B24">
<label>24</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Mayne</surname> <given-names>E</given-names>
</name>
<name>
<surname>Funderburg</surname> <given-names>N</given-names>
</name>
<name>
<surname>Sieg</surname> <given-names>S</given-names>
</name>
<name>
<surname>Asaad</surname> <given-names>R</given-names>
</name>
<name>
<surname>Kalinowska</surname> <given-names>M</given-names>
</name>
<name>
<surname>Rodriguez</surname> <given-names>B</given-names>
</name>
<etal/>
</person-group>. <article-title>Increased platelet and microparticle activation in HIV infection: upregulation of P-selectin and tissue factor expression</article-title>. <source>J acquired Immune deficiency syndromes (1999)</source>. (<year>2012</year>) <volume>59</volume>:<page-range>340&#x2013;6</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1097/QAI.0b013e3182439355</pub-id>
</citation>
</ref>
<ref id="B25">
<label>25</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Hijmans</surname> <given-names>J</given-names>
</name>
<name>
<surname>Stockelman</surname> <given-names>K</given-names>
</name>
<name>
<surname>Garcia</surname> <given-names>V</given-names>
</name>
<name>
<surname>Levy</surname> <given-names>M</given-names>
</name>
<name>
<surname>Brewster</surname> <given-names>L</given-names>
</name>
<name>
<surname>Bammert</surname> <given-names>T</given-names>
</name>
<etal/>
</person-group>. <article-title>Circulating microparticles are elevated in treated HIV -1 infection and are deleterious to endothelial cell function</article-title>. <source>J Am Heart Assoc</source>. (<year>2019</year>) <volume>8</volume>:<fpage>e011134</fpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.1161/JAHA.118.011134</pub-id>
</citation>
</ref>
<ref id="B26">
<label>26</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Marques de Menezes</surname> <given-names>E</given-names>
</name>
<name>
<surname>Deng</surname> <given-names>X</given-names>
</name>
<name>
<surname>Liu</surname> <given-names>J</given-names>
</name>
<name>
<surname>Bowler</surname> <given-names>S</given-names>
</name>
<name>
<surname>Shikuma</surname> <given-names>C</given-names>
</name>
<name>
<surname>Stone</surname> <given-names>M</given-names>
</name>
<etal/>
</person-group>. <article-title>Plasma CD16 + Extracellular vesicles associate with carotid artery intima-media thickness in HIV + Adults on combination antiretroviral therapy</article-title>. <source>mBio</source>. (<year>2022</year>) <volume>13</volume>. doi:&#xa0;<pub-id pub-id-type="doi">10.1128/mbio.03005-21</pub-id>
</citation>
</ref>
<ref id="B27">
<label>27</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Sadallah</surname> <given-names>S</given-names>
</name>
<name>
<surname>Amicarella</surname> <given-names>F</given-names>
</name>
<name>
<surname>Eken</surname> <given-names>C</given-names>
</name>
<name>
<surname>Iezzi</surname> <given-names>G</given-names>
</name>
<name>
<surname>Schifferli</surname> <given-names>J</given-names>
</name>
</person-group>. <article-title>Ectosomes released by platelets induce differentiation of CD4+T cells into T regulatory cells</article-title>. <source>Thromb haemostasis</source>. (<year>2014</year>) <volume>112</volume>:<page-range>1219&#x2013;29</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1160/TH14-03-0281</pub-id>
</citation>
</ref>
<ref id="B28">
<label>28</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Marques de Menezes</surname> <given-names>E</given-names>
</name>
<name>
<surname>Ramallho</surname> <given-names>J</given-names>
</name>
<name>
<surname>Bucovsky</surname> <given-names>M</given-names>
</name>
<name>
<surname>Shane</surname> <given-names>E</given-names>
</name>
<name>
<surname>Yin</surname> <given-names>M</given-names>
</name>
<name>
<surname>Norris</surname> <given-names>P</given-names>
</name>
</person-group>. <article-title>Serum extracellular vesicles expressing bone activity markers associate with bone loss after HIV antiretroviral therapy</article-title>. <source>AIDS (London England)</source>. (<year>2020</year>) <volume>34</volume>:<page-range>351&#x2013;61</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1097/QAD.0000000000002430</pub-id>
</citation>
</ref>
<ref id="B29">
<label>29</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Pannetier</surname> <given-names>L</given-names>
</name>
<name>
<surname>Tamagne</surname> <given-names>M</given-names>
</name>
<name>
<surname>Bocquet</surname> <given-names>T</given-names>
</name>
<name>
<surname>Pirenne</surname> <given-names>F</given-names>
</name>
<name>
<surname>Ansart-Pirenne</surname> <given-names>H</given-names>
</name>
<name>
<surname>Vingert</surname> <given-names>B</given-names>
</name>
</person-group>. <article-title>HLA molecule expression on the surface of cells and microparticles in platelet concentrates</article-title>. <source>Transfusion</source>. (<year>2021</year>) <volume>61</volume>:<page-range>1023&#x2013;8</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1111/trf.16201</pub-id>
</citation>
</ref>
<ref id="B30">
<label>30</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Yin</surname> <given-names>Y</given-names>
</name>
<name>
<surname>Chen</surname> <given-names>H</given-names>
</name>
<name>
<surname>Wang</surname> <given-names>Y</given-names>
</name>
<name>
<surname>Zhang</surname> <given-names>L</given-names>
</name>
<name>
<surname>Wang</surname> <given-names>X</given-names>
</name>
</person-group>. <article-title>Roles of extracellular vesicles in the aging microenvironment and age-related diseases</article-title>. <source>J extracellular vesicles</source>. (<year>2021</year>) <volume>10</volume>:<fpage>e12154</fpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.1002/jev2.12154</pub-id>
</citation>
</ref>
<ref id="B31">
<label>31</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Damien</surname> <given-names>P</given-names>
</name>
<name>
<surname>Cognasse</surname> <given-names>F</given-names>
</name>
<name>
<surname>Lucht</surname> <given-names>F</given-names>
</name>
<name>
<surname>Suy</surname> <given-names>F</given-names>
</name>
<name>
<surname>Pozzetto</surname> <given-names>B</given-names>
</name>
<name>
<surname>Garraud</surname> <given-names>O</given-names>
</name>
<etal/>
</person-group>. <article-title>Highly active antiretroviral therapy alters inflammation linked to platelet cytokines in HIV-1-infected patients</article-title>. <source>J Infect diseases</source>. (<year>2013</year>) <volume>208</volume>:<page-range>868&#x2013;70</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1093/infdis/jit260</pub-id>
</citation>
</ref>
<ref id="B32">
<label>32</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Peltenburg</surname> <given-names>N</given-names>
</name>
<name>
<surname>Bierau</surname> <given-names>J</given-names>
</name>
<name>
<surname>Bakker</surname> <given-names>J</given-names>
</name>
<name>
<surname>Schippers</surname> <given-names>J</given-names>
</name>
<name>
<surname>Lowe</surname> <given-names>S</given-names>
</name>
<name>
<surname>Paulussen</surname> <given-names>A</given-names>
</name>
<etal/>
</person-group>. <article-title>Erythrocyte Inosine triphosphatase activity: A potential biomarker for adverse events during combination antiretroviral therapy for HIV</article-title>. <source>PloS One</source>. (<year>2018</year>) <volume>13</volume>:<fpage>e0191069</fpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.1371/journal.pone.0191069</pub-id>
</citation>
</ref>
<ref id="B33">
<label>33</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Peltenburg</surname> <given-names>N</given-names>
</name>
<name>
<surname>Bierau</surname> <given-names>J</given-names>
</name>
<name>
<surname>Schippers</surname> <given-names>J</given-names>
</name>
<name>
<surname>Lowe</surname> <given-names>S</given-names>
</name>
<name>
<surname>Paulussen</surname> <given-names>A</given-names>
</name>
<name>
<surname>van den Bosch</surname> <given-names>B</given-names>
</name>
<etal/>
</person-group>. <article-title>Metabolic events in HIV-infected patients using abacavir are associated with erythrocyte inosine triphosphatase activity</article-title>. <source>J antimicrobial chemotherapy</source>. (<year>2019</year>) <volume>74</volume>:<page-range>157&#x2013;64</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1093/jac/dky383</pub-id>
</citation>
</ref>
<ref id="B34">
<label>34</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Latham</surname> <given-names>V</given-names>
</name>
<name>
<surname>Stebbing</surname> <given-names>J</given-names>
</name>
<name>
<surname>Mandalia</surname> <given-names>S</given-names>
</name>
<name>
<surname>Michailidis</surname> <given-names>C</given-names>
</name>
<name>
<surname>Davies</surname> <given-names>E</given-names>
</name>
<name>
<surname>Bower</surname> <given-names>M</given-names>
</name>
<etal/>
</person-group>. <article-title>Adherence to trizivir and tenofovir as a simplified salvage regimen is associated with suppression of viraemia and a decreased cholesterol</article-title>. <source>J antimicrobial chemotherapy</source>. (<year>2005</year>) <volume>56</volume>:<page-range>186&#x2013;9</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1093/jac/dki170</pub-id>
</citation>
</ref>
<ref id="B35">
<label>35</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Durand-Gasselin</surname> <given-names>L</given-names>
</name>
<name>
<surname>Da Silva</surname> <given-names>D</given-names>
</name>
<name>
<surname>Benech</surname> <given-names>H</given-names>
</name>
<name>
<surname>Pruvost</surname> <given-names>A</given-names>
</name>
<name>
<surname>Grassi</surname> <given-names>J</given-names>
</name>
</person-group>. <article-title>Evidence and possible consequences of the phosphorylation of nucleoside reverse transcriptase inhibitors in human red blood cells</article-title>. <source>Antimicrobial Agents chemotherapy</source>. (<year>2007</year>) <volume>51</volume>:<page-range>21055&#x2013;2111</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1128/AAC.00831-06</pub-id>
</citation>
</ref>
<ref id="B36">
<label>36</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Al Mamun Bhuyan</surname> <given-names>A</given-names>
</name>
<name>
<surname>Signoretto</surname> <given-names>E</given-names>
</name>
<name>
<surname>Bissinger</surname> <given-names>R</given-names>
</name>
<name>
<surname>Lang</surname> <given-names>F</given-names>
</name>
</person-group>. <article-title>Enhanced eryptosis following exposure to dolutegravir</article-title>. <source>Cell Physiol Biochem</source>. (<year>2016</year>) <volume>39</volume>:<page-range>639&#x2013;50</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1159/000445655</pub-id>
</citation>
</ref>
<ref id="B37">
<label>37</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Wang</surname> <given-names>W</given-names>
</name>
<name>
<surname>Mao</surname> <given-names>L</given-names>
</name>
<name>
<surname>Lai</surname> <given-names>H</given-names>
</name>
<name>
<surname>Wang</surname> <given-names>Y</given-names>
</name>
<name>
<surname>Jiang</surname> <given-names>Z</given-names>
</name>
<name>
<surname>Li</surname> <given-names>W</given-names>
</name>
<etal/>
</person-group>. <article-title>Dolutegravir derivative inhibits proliferation and induces apoptosis of non-small cell lung cancer cells via calcium signaling pathway</article-title>. <source>Pharmacol Res</source>. (<year>2020</year>) <volume>161</volume>:<fpage>105129</fpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.1016/j.phrs.2020.105129</pub-id>
</citation>
</ref>
<ref id="B38">
<label>38</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Theron</surname> <given-names>A</given-names>
</name>
<name>
<surname>Anderson</surname> <given-names>R</given-names>
</name>
<name>
<surname>Madzime</surname> <given-names>M</given-names>
</name>
<name>
<surname>Rossouw</surname> <given-names>T</given-names>
</name>
<name>
<surname>Steel</surname> <given-names>H</given-names>
</name>
<name>
<surname>Meyer</surname> <given-names>P</given-names>
</name>
<etal/>
</person-group>. <article-title>Pro-inflammatory interactions of dolutegravir with human neutrophils in an <italic>in vitro</italic> study</article-title>. <source>Molecules (Basel Switzerland)</source>. (<year>2022</year>) <volume>27</volume>:<fpage>9057</fpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.3390/molecules27249057</pub-id>
</citation>
</ref>
<ref id="B39">
<label>39</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Madzime</surname> <given-names>M</given-names>
</name>
<name>
<surname>Theron</surname> <given-names>A</given-names>
</name>
<name>
<surname>Anderson</surname> <given-names>R</given-names>
</name>
<name>
<surname>Tintinger</surname> <given-names>G</given-names>
</name>
<name>
<surname>Steel</surname> <given-names>H</given-names>
</name>
<name>
<surname>Meyer</surname> <given-names>P</given-names>
</name>
<etal/>
</person-group>. <article-title>Dolutegravir potentiates platelet activation by a calcium-dependent, ionophore-like mechanism</article-title>. <source>J immunotoxicology</source>. (<year>2022</year>) <volume>19</volume>:<fpage>1</fpage>&#x2013;<lpage>8</lpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.1080/1547691X.2022.2142705</pub-id>
</citation>
</ref>
</ref-list>
</back>
</article>
