There are two types of extracellular vesicle (EVs): exosomes, which are small vesicles (40-100 nm, derived from intracellular membrane compartments), and larger extracellular vesicles (300-900 nm in diameter). The largest extracellular vesicles, also known as microparticles or ectosomes, are generated by the budding of the cell membrane; they play a major role in intercellular communication under physiological and pathological conditions (1). In this study, we focused exclusively on these larger vesicles budding from the plasma membrane of cells, which we will refer to hereafter as EVs. Most studies on EVs have shown an immunoregulatory role for heterologous EVs in transfusion models (2–6). By contrast, the role of the EVs naturally present in blood remains unclear (3). However, these autologous EVs can also be associated with disease. Indeed, the immunoregulatory properties of EVs have been highlighted in autoimmune diseases (1–7).

Blood EVs have diverse cellular origins and their numbers are highly variable; many factor contribute to this variability (1, 2). These variations affect immune system activation, as shown, particularly, for the activation and differentiation of CD4⁺ T lymphocytes (TLs) induced by heterologous EVs (2–5). The variability of autologous EVs may play a similar role under physiological conditions.

We tested this hypothesis, by investigating the functionality of autologous EVs with CD4⁺ TLs. We treated purified CD4⁺ TLs from healthy donors (HDs) with different concentrations of EVs. This dose-effect assay covered the range of variability of the number of EVs observed under physiological conditions (2). We explored the expression of several immunoregulatory molecules on the cell surface (OX40/OX40L, CD40/CD40L, PD1 and ICOS) and in the culture supernatant: CD357 (GITR), CD270 (HVEM), IDO, CD30, CD40, DR6, CD95 (Fas), CD120a/b (TNF-R1 and -R2), TRAIL-R1/-R2, CD272 (BTLA), CD223 (LAG-3), PD1/PD-ligands, CD366 (Tim-3), CD28/CD80, CD137 (4-1BB), CD27 and CD152 (CTLA-4). All these molecules were selected on the basis of major roles they play in immune system regulation or their possible association with EVs (1, 2, 8–12). We also investigated whether autologous EVs had the same lymphoproliferative properties as heterologous EVs in response to polyclonal or specific-antigen stimulation (2, 3).

We mimicked the conditions in the bloodstream in vivo as closely as possible, by studying the autologous blood EVs present in plasma directly, without ultracentrifugation. The results obtained with this direct phenotyping method were similar to published results, particularly for the phenotyping of EVs in HDs (2, 3, 14).. This direct plasma EV phenotyping also revealed heterogeneity in the numbers and size distribution of EVs ( Supplemental Table 1 ).

Our objective here was to determine whether autologous EVs play a direct role in regulating the immune system, similar to that played by heterologous EVs. The data obtained in this in vitro study suggest that, regardless of treatment time, autologous EVs have no direct effect on CD4⁺ TL activation.

However, EVs are involved in intercellular communication (3–5). The absence of variation in the expression of the major immunoregulatory molecules studied suggested a possible indirect action of EVs on autologous CD4⁺ TLs ( Figure 1 ). The detection of sCD27 is also consistent with such an indirect effect. sCD27 is an alternative form of the CD27 transmembrane receptor. sCD27 has been detected in the biological fluids of HDs and patients with various diseases (12, 15–18). Conflicting findings have been published concerning sCD27, but most studies have reported that this protein contributes to cell activation and lymphoproliferation following the triggering of the TcR/CD3 complex (12, 19–22). For CD4⁺ TLs, sCD27, in association with its CD70 ligand, has been shown to upregulate the expression of CD40L and to stimulate lymphoproliferation (12, 20, 23). However, no change in CD40L levels was detected in this study ( Figure 1 ). Even though this phenotypic change was not observed with these autologous EVs, CD40/CD40L was reported to play a role for a certain type of EV in a previous study (3). Nevertheless, we still do not know whether CD40/CD40L plays an important role in the situation studied here, these molecules being present on EVs or CD4⁺ TLs (2).

However, sCD27 may also be provided by CD27⁺ EVs during culture. Indeed, the presence of CD27⁺ EVs has recently been reported in the plasma of HIV-infected patients (14). We purified CD27⁺ EVs by flow cytometry to test this hypothesis. CD27⁺ EVs were tested with the same Luminex assays used to detect sCD27. We detected no purified CD27⁺ EVs in Luminex assays ( Supplemental Figure 2B ). The sCD27 detected in supernatants cannot, therefore, have originated from the CD27⁺ EVs initially added to the culture.

The reason for the induction of sCD27 by CD4⁺ TLs in response to EVs remains unclear, but it may have consequences for lymphoproliferation. Nevertheless, we followed up on the sCD27 lead and the work of Huang J. et al., by investigating whether sCD27 could promote the lymphoproliferation of stimulated T cells (12). We exposed T cells to polyclonal activation with SEB or antigen-specific PPD restimulation. Once the TcR was committed in response to such stimulation, EVs significantly increased the proliferation of T cells ( Figure 3 ). Our data with SEB confirm previously reported data obtained by polyclonal PHA activation (3). However, this previous study did not take the dose effect into account (3), potentially accounting for the lack of a proliferative effect in the absence of mitogen treatment (3). Nevertheless, our results for the PPD vaccinal antigen confirm that autologous EVs facilitate the lymphoproliferation of specific CD4⁺ TLs.

Our data provide evidence of a mechanism of interaction between autologous EVs and CD4⁺ TLs different from that reported for heterologous EVs supplied by transfusion (2). Indeed, unlike heterologous EVs (2), autologous EVs cannot induce conventional CD4⁺ TL proliferation without prior involvement of the TcR. However, Treg, but also circulating Tfh, Th17 or other subpopulations of CD4⁺ TLs play major roles in inducing or controlling adaptive immune responses, and heterologous EVs have been shown to be functional on these cells (2, 4–6). Finally, we investigated whether autologous EVs could have a functional effect on autologous cells at higher concentrations in the blood, by performing functional lymphoproliferation tests on Tfh, Treg and Th17 cells. However, as with conventional CD4⁺ TLs, autologous EVs did not induce the proliferation of these subpopulations ( Supplemental Figure 3 ).

Heterologous EVs have been shown to induce lymphoproliferation at low concentrations of between 20,000:1 and 200:1 (2). Studies were performed with additional ratios (200 and 2000:1 with 200,000 cells in culture), but no lymphoproliferation was detected with autologous EVs at any of these ratios (data not shown).

The cellular origin of EVs might underlie functional modifications of the immune system. Several groups have reported functional differences between EVs of different cellular origins in heterologous transfusion studies (3–6). However, these studies did not specifically purify these EVs. EVs from apheresis-derived platelet concentrates or from red blood cell concentrates do not consist exclusively of platelet-EVs or erythrocyte-EVs (2, 3). Nevertheless, studies of EVs from stored blood concentrates have provided an indication as to the potential role of some EVs (3). Indeed, during blood concentrate storage, a change occurs in the source of the EVs present, and a particular type of EVs, such as erythrocyte-EVs, may come to predominate (3).

Moreover, we cannot exclude the possibility that specific autologous EVs with costimulatory molecules are associated with lymphoproliferation after TCR engagement, as already reported for heterologous EVs (2).

EVs naturally present in the blood may play a functional role in intercellular communication in the immune system, by promoting lymphoproliferation after TcR engagement, probably with the assistance of sCD27 ( Figure 4 ). However, further studies are required to elucidate the link between sCD27 secretion by autologous CD4⁺ TLs and EVs.

Finally, these autologous EVs may, therefore, play a key role in pathological conditions, particularly in patients in which EV levels are high due to infection, autoimmune disease, or cancer (1, 24–27). It will also be important to take these findings into account in cases of autotransfusion for pathological conditions or in the case of doping practices (28, 29).

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study.

BV was the principal investigator and takes primary responsibility for the paper; SP was the clinical research associate for this study. DN-L, SL and MT performed the laboratory work. DN-L and BV analyzed the results; BV and FP coordinated the research; DN-L and BV wrote the paper; FP, LC and AM reviewed the paper. All the authors contributed to the manuscript and approved the submitted version.

This work was supported by the scientific committee of Agence Française de Lutte contre le Dopage (AFLD), the World Antidoping Agency (WADA), Etablissement Français du Sang, INSERM and Université Paris-Est Créteil. We are particularly grateful to the healthy blood donors who participated in this study, and the EFS team responsible for collecting blood donations. We would like to thank Muriel Andrieu and Souganya Many from the flow cytometry facility of the Cochin Institute (CYBIO Core facility).

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.

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.