Circulating exosomes express α4β7 integrin and compete with CD4+ T cells for the binding to Vedolizumab

Rossana Domenis; Marco Marino; Adriana Cifù; Giulia Scardino; Francesco Curcio; Martina Fabris

doi:10.1371/journal.pone.0242342

. 2020 Nov 12;15(11):e0242342. doi: 10.1371/journal.pone.0242342

Circulating exosomes express α₄β₇ integrin and compete with CD4+ T cells for the binding to Vedolizumab

Rossana Domenis ¹, Marco Marino ², Adriana Cifù ¹, Giulia Scardino ², Francesco Curcio ^1,³, Martina Fabris ^3,^*

Editor: Aftab A Ansari⁴

PMCID: PMC7661055 PMID: 33180848

Abstract

Vedolizumab (VDZ) is a therapeutic monoclonal antibody approved for the treatment of inflammatory bowel diseases (IBD). VDZ selectively binds to the α4β7 integrin and blocks trafficking of a specific subset of gastrointestinal-homing T-lymphocytes to inflamed tissue. Although VDZ has shown promising results in numerous clinical studies a subgroup of patients do not respond adequately. Mechanistic insights and prognostic biomarkers able to predict which patients might benefit from VDZ therapy are currently lacking. Circulating exosomes were isolated from serum of blood donors and VDZ-treated patients by polymer-based precipitation. The surface expression of α4β7 integrin was evaluated by flow cytometry and the levels of exosome-bound VDZ were investigated by Promonitor-VDZ ELISA kit. The capacity of exosomes to interfere with the adhesion of VDZ-treated CD4+ T cells was assessed by adhesion assay. In this study, we showed that serum exosomes isolated from both blood donor and ulcerative colitis patients express on their surface the VDZ target α4β7 integrin. We observed an increased exosomal sequestration of VDZ in anti-TNF exposed patients compared to anti- TNFα naïve patients, according to a greater expression of α4β7 integrin on vesicles surface. Circulating exosomes could compete for VDZ binding with CD4+ T cells since we found that the amount of VDZ bound to T cells was impaired in the presence of exosomes. In addition, we demonstrated that exosomes bind VDZ, which consequently becomes unable to block MadCAM-1-mediated adhesion of lymphocytes. Circulating exosomes might contribute to drug sequestration, possibly affecting the therapeutic efficacy of VDZ in IBD patients. Our data suggest that previous biologic therapy may have altered the sequestration capacity of circulating exosomes, thus reducing the efficacy of VDZ in patients who failed anti-TNF agents.

Introduction

Inflammatory bowel diseases (IBD) are a chronic and highly heterogeneous group of diseases, including Crohn’s disease (CD) and ulcerative colitis (UC), which are characterized by inflammation of the intestinal tract. Although the exact etiopathogenic mechanism of IBD remains unknown, accumulating evidence suggests that they result from an inappropriate inflammatory response to different intestinal antigens in a genetically susceptible host [1].

The increased knowledge of the pathways involved in IBD pathogenesis has led to the development of new treatment options. Biologic agents, such as monoclonal antibodies against specific cytokines or receptors, represent a class of therapeutic molecules with the capacity to selectively block the major proinflammatory cascades involved in IBD and, consequently, the activation and proliferation of intestinal T lymphocytes, therefore reestablishing the balance between pro- and anti-inflammatory stimuli [2].

The infiltration of leukocytes in the gut mucosa is mediated in part by interactions between integrins expressed on lymphocytes, and their ligands MAdCAM-1 on the gut epithelial cells [3]. Vedolizumab (VDZ) is a humanized IgG1 monoclonal antibody that binds specifically to the α₄β₇ integrin expressed on a subset of primed gut-homing T-lymphocytes [4]. The inhibition of lymphocytes migration selectively downregulates gut inflammation, while preserving systemic immune responses.

Although VDZ is successfully used for treatment of IBD naïve patients or for patients who have failed TNF-α antagonists [5], a significant proportion of patients does not respond at induction or loses response over time, and might develop significant side effects [6–8]. Primary or secondary loss of response to biological therapy may be caused by several potential mechanisms: advanced disease, inadequate dosing, development of neutralizing anti-drug antibodies (ADA), disease heterogeneity/cytokine switch, metabolism, accelerated clearance [9]. Clearly, a better understanding of the factors associated with drug response may allow early identification of patients who are most likely to benefit from VDZ or any other biologic agent [10].

Exosomes are a population of bilayer nanometer-sized extracellular vesicles that originate from the multivescicular endosome and are secreted by several types of living cells in all body fluids. They are regarded as major players in cell-cell communication as they transfer specific lipid, nucleic acids, proteins and other bioactive molecules from the parental cells to the neighboring or distant recipient cells [11]. In addition to their physiological functions, exosomes are involved in the pathological development and progression of numerous diseases. Recently, it has been proposed that they have a role in the pathogenesis of diverse inflammatory conditions, including cancer, type 2 diabetes, obesity, rheumatoid arthritis and neurodegenerative diseases, creating a microenvironment that triggers inflammation and sustains disease progression [12].

To date there are few studies examining the role of exosomes in IBD. Extracellular vesicles isolated from luminal fluid of IBD patients exhibit a proinflammatory effect on epithelial cells and macrophages, inducing the release of the pro-inflammatory cytokine IL-8 [13]. In addition, exosomes released by intestinal epithelial cells and naïve macrophages upon CD-associated adherent-invasive Escherichia coli infection can trigger and amplify proinflammatory responses [14]. Moreover, serum-derived exosomes in a murine model of induced acute colitis, transport acute-phase proteins and immunoglobulins that are mainly involved in the complement and coagulation cascade [15].

The content of exosomes is cell type specific and reflects the phenotypic state of the parental cell, hence exosomes are considered "fingerprints" of the releasing cell. Therefore, they could be potentially used as disease specific markers. For example, it has been reported that the level of salivary exosomal proteasome subunit alpha type 7 (PSMA7) differs significantly between healthy individuals and IBD patients and may reflect early disease [16]. It has also been reported that patients with IBD secrete an increased number of annexin-1-containing exosomes in the blood stream, possibly released by intestinal epithelial cells to help restore damaged mucosa [17].

Interestingly, it has been proposed that exosomes could also be involved in mediating drug resistance by sequestration of therapeutic molecules into the vesicles. Specifically, exosomes secreted by breast cancer patients’ serum, carrying HER2 antigen that is the target of Trastuzumab, contribute to drug sequestration, reducing therapeutic efficacy [18].

In this study, we characterized exosomes isolated from serum of ulcerative colitis patients treated with VDZ in order to address the possibility that circulating exosomes bind VDZ and interfere with its therapeutic efficacy.

Methods

Study population

This single-centre retrospective study was approved by the Institutional Review Board of the Department of Medical Area (IRB-DAME), University of Udine (10/IRB_CURCIO_2019). The study included 17 ulcerative colitis (UC) patients (mean age 40.7 ± 17.5 years, 50% female), 6 naïve to biologics and 11 previously treated with at least one anti-TNFα agent (Infliximab n = 2, Adalimumab n = 3 or both n = 6), starting Vedolizumab (VDZ) therapy and followed at the Gastroenterology Unit of the University Hospital of Udine. Baseline patient details including disease duration, smoking status, concurrent medications and Mayo score were collected. Demographic and disease characteristics of the patients at baseline were listed in Table 1 (failure to TNF-α) and in Table 2 (naïve to TNF-α). All the subjects at the beginning of VDZ therapy had confirmed moderately to severely active ulcerative colitis, defined as a total score of 6 to 12 on the Mayo scale (total Mayo scores range from 0 to 12, with higher scores indicating more severe disease) and a subscore between 2 or 3 on the endoscopic component of the Mayo scale. Of the 11 subjects previously treated with at least one anti-TNFα agent, 6 interrupted the treatment for primary failure, 2 for secondary failure and 3 for adverse effects. Enrolled patients had been treated with VDZ for at least 2 months (range 2 to 18 months). Peripheral blood samples were collected immediately before VDZ infusion and centrifuged immediately at 2500g for 10 min to obtain the serum which was stored in aliquots at -80°C until analysis. Sera from 12 age and sex-matched healthy adults (mean age 41 ± 16.5 years, 50% female) were used as controls. This retrospective study was approved by the local Institutional Review Board (10/IRB_CURCIO_2019).

Table 1. Baseline characteristics of the IBD patients anti TNF-α failure.

Pt	Age/Sex	Lenght Disease (yrs)	Mayo score Clin/End	Smokers (Y/N)	TNF-α drugs	TNF-α Failure PF/SF/AE	VDZ Resp. R/PF/SF
1	M/49	5 to 10	12/3	N	IFX	PF	PF
2	M/59	> 10	6/3	Y	ADL	PF	R
3	M/54	5 to 10	8/2	N	ADL/IFX	PF	R
4	M/41	< 5	9/3	N	IFX	AE	R
5	M/31	5 to 10	7/2	N	ADL/IFX	SF	SF
6	F/22	< 5	8/2	N	ADL	AE	SF
7	F/24	5 to 10	8/3	N	ADL/IFX	PF	SF
8	F/36	5 to 10	7/3	N	ADL/IFX	SF	R
9	F/31	< 5	8/3	N	ADL	PF	R
10	F/39	< 5	8/2	N	ADL/IFX	AE	R
11	M/19	< 5	7/3	N	ADL/IFX	PF	SF

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IFX = infliximab, GOL = golimumab, ADL = adalimumab, R = responder, PF = primary failure, SF = secondary failure, AE = adverse event.

Table 2. Baseline characteristics of the IBD patients naïve to TNF-α.

Pt	Age/Sex	Lenght Disease (yrs)	Mayo score Clin/End	Smokers (Y/N)	VDZ Resp. R/PF/SF
1	M/74	5 to 10	7/3	N	R
2	F/68	5 to 10	8/2	N	SF
3	F/35	< 5	9/3	N	R
4	F/19	< 5	8/3	N	R
5	F/43	> 10	7/2	N	SF
6	M/23	< 5	9/3	N	R

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R = responder, PF = primary failure, SF = secondary failure.

Exosome isolation

Exosomes were isolated from the serum of patients and blood donors (CTRL) with three different technologies. Serum (250 μL) was centrifuged at 3000g for 15 minutes, filtered with 0.22 μm filter and concentrated in Amicon^® Ultra-4 (100 kDa). Samples were incubated with ExoQuick (System Biosciences) for 30 minutes at 4°C and then centrifuged twice at 1500g for 30 and 5 minutes, respectively. The exosome-containing pellet was then resuspended in 250 μL of PBS buffer and stored -20°C for subsequent analysis. Alternatively, 2 ml of serum was overlaid on qEV2 size exclusion column (Izon) followed by elution with PBS, according to manufacturer’s protocol. The fractions contained exosomes were pooled, filtered with 0.22 μm filter and concentrated in Amicon^® Ultra-4 (100 kDa) to a final volume of 150 μL. Finally, exosomes were extracted by exoEasy Maxi Kit (Qiagen), according to the manufacturer′s protocol. Briefly, 4 ml of pre-filtered serum was mixed with an equal volume of buffer XBP, transferred into the exoEasy spin column and centrifuged at 500g for 1 min. The flow-through was discarded and the spin column was washed with 10 mL buffer XWP. Finally, 400 μL of buffer XE was added to membrane and spin column was centrifuged at 5000g for 5 min to elute the exosomes.

Exosome characterization

The number of isolated exosomes was determined using the Exocet kit (System Biosciences), according to manufacturer’s protocol [19].

Exosomes were analyzed for the expression of exosomal markers and proteins expressed on immune cells surface by flow cytometry and immunoblotting [20]. Specifically, vesicles (3x10⁹) purified by immunoaffinity Exo-Flow kit (System Biosciences) were stained with specific monoclonal antibodies anti-CD81 FITC (Biolegend), anti-CD63 FITC (Santa Cruz), anti-CD9 PE (eBiosciences), anti-CD3 APC (eBiosciences), anti-CD14 PE (eBiosciences), anti-MadCAM-1 FITC (LSBio) and anti-integrin α₄β₇ FITC (Biorbyt). The flow cytometry analysis was performed calculating the percentage of exosome-bound beads compared with beads alone. Isolated exosomes (50 μg for exosomes isolated by Exoquick and exoEasy and 25 μg for exosomes isolated by qEv2) were boiled in Laemmli sample buffer and subjected to electrophoresis on Mini-PROTEAN TGX precast 10% gels (BioRad). Primary antibodies against flotillin-2 (1:1000, Cell Signaling), CD9-HRP conjugated (1:500, Novus biologicals) and TSG101-HRP conjugated (1:1000, Santa Cruz) were used for immunoblotting analysis. Exosomes were analyzed by nanoparticle tracking analysis (NTA), using the NanoSight LM10 system (Malvern). For each sample, diluted in PBS (1000–10,000 times), a video was captured for 60s each with a detection threshold set at 16 (maximum).

The Promonitor^®-VDZ ELISA test (Progenika Biopharma-Grifols) was used to quantify the levels of VDZ in patient’s serum and exosome samples, according to protocol from the manufacturer. Monitoring of ADA against TNF-α and VDZ was performed utilizing non-cross reactive ADA detection ELISA kits from Progenika Biopharma-Grifols according to the manufacturer’s instruction. None of the sera showed detectable levels of ADA.

Analysis of VDZ binding to exosomes

Exosomes isolated from a pool of blood donor’s serum were incubated with increasing amount of VDZ (from 6.3 to 100 μg) for 2h at 4°C on orbital shaker and purified again by Exoquick.

The levels of exosome-bound VDZ were investigated by western blot analysis. Purified exosomes were lysed in RIPA buffer and 20 μg of proteins were loaded for polyacrylamide native gel electrophoresis, then transferred to PVDF membranes for blocking and subsequent probing with primary antibodies against VDZ-HRP conjugated (1:1000, Progenika Biopharma-Grifols). Signals were detected using chemiluminescence method (SuperSignal^™ West Femto substrate, Thermo Fischer Scientific).

Analysis of exosome competition for binding to VDZ

Exosomes and CD4+ T cells purified from 1ml of blood donor serum were incubated alone or together with 60 μg of VDZ for 1h at 4°C on orbital shaker, then cells were washed twice with PBS and lysed in RIPA buffer in ice for 30’. Protein extracted were separated by native electrophoresis and probed with primary antibodies against VDZ-HRP conjugated.

MAdCAM adhesion assay

High-binding polystyrene 96-well plates were coated (100 μl/well) with 2.5 μg/mL of Recombinant Human MAdCAM-1 Fc Chimera (R&D system) diluted in PBS and incubated overnight at 37°C. Nonspecific binding sites were blocked with 1% BSA in PBS w/Ca²⁺/Mg²⁺ for 1h at 37°C. CD4+ T cells (4x10⁵) were preincubated in binding buffer (1mM MnCl₂ and 1% BSA in PBS w/Ca²⁺/Mg²) with different concentration of VDZ (50 μg/mL, 5 μg/mL or 5 ng/mL) without or with 5x10⁹ exosomes for 15 min. Then, cells were added to MAdCAM-1-coated plate and incubated for 90 min at 37°C. Adherent cells were stained with Hoechst dye and visualized by fluorescence microscopy (Leica DMI 6000B) coupled to a CCD camera (Leica DFC350FX).

Statistical analysis

Data are expressed as the mean ± standard deviation. Unpaired t or Mann-Withney test was used to compare CTRL vs UC patients and anti- TNFα naïve vs anti-TNFα antagonist-experienced patients. p < 0.05 was considered statistically significant.

Results

Purification and characterization of serum exosomes from patients and controls

As illustrated in Fig 1A, the mean concentration of exosomes isolated from sera of UC patients was lower than in CTRL, even though the difference was not significant (1.3x10¹¹ ±8.5x10¹⁰ versus 7.1x10¹⁰ ±3.8x10¹⁰, p = 0.058). By contrast, exosomal concentration was greater in patients previously exposed to anti-TNFα agents compared to those naïve to biologics (4.9x10¹¹±2.3x10¹⁰ versus 8.3x10¹⁰ ±40x10¹⁰, p = 0.047) (Fig 1B).

Flow cytometry analyses demonstrated that exosomes isolated from UC patients and CTRL expressed the expected surface tetraspanins, such as the CD9, CD63 and CD81 and were positive for exosome stain Exo-FITC, a fluorescent dye that recognizes post-translational modifications on exosomal surface proteins (see Fig 1C as an example).

Serum-derived exosomes expressed the α₄β₇ integrin and were able to bound VDZ

The expression of exosomal marker CD9 (Fig 2B), T cells marker CD3 (Fig 2C) and monocytes marker CD14 (Fig 2D) was not different in exosomes isolated from UC patients compared with CTRL and from anti-TNFα-antagonist exposed patients compared to anti- TNFα naïve patients. Of note, serum-derived exosomes expressed the VDZ target α4β7 integrin (Fig 2E), but not its ligand, the MAdCAM-1 protein (Fig 2F). Exosomes from UC patients expressed more integrin α₄β₇ than CTRL, however the difference was not statistically significant (16 ±12.4 versus 22.8±17.9, p = 0.097), whereas the expression of α4β7 integrin in exosomes of TNFα-antagonist exposed patients was greater than in anti- TNFα naïve patients (14.1 ±4.1 versus 27.6±20.8, p = 0.047).

To verify the hypothesis that circulating exosomes of UC patients expressing the α₄β₇ integrin may carry VDZ in the follow-up, the amount of VDZ in serum, free and bound to exosomes, was measured by ELISA. Fig 3A shows that VDZ was measurable in serum both as free molecule and bound to exosomes. The expression of α₄β₇ integrin and the presence of VDZ bound to UC exosomes was also confirmed by native immunoblotting (Fig 3B). As expected, exosomes isolated from serum of CTRL patient was negative for VDZ as they are not under therapy, while the signal was present in UC patients.

Considering the amount of VDZ bound by exosomes as a percentage of the total VDZ measured in serum (Fig 3C), we observed an increased sequestration of the medication in anti-TNFα -antagonist exposed compared to anti- TNFα naïve patients (61.5 ±26% versus 36.7 ±16.8%; p = 0.037). Finally, there was a correlation (trending towards significance) between the levels of α₄β₇ integrin and the amounts of VDZ bound to exosomes in UC patients (Fig 3D).

Finally, to confirm that VDZ is bound to exosomes and does not precipitate as a contaminant, we compared exosomes isolated from a pool of UC patients sera (n = 5) by Exoquick with other two commercial kits (ExoEasy and qEV2). The concentration of exosomes varied in the samples isolated using the different methods and polymer-precipitation based method ensured the highest yield (Fig 4A). Immunoblotting analysis confirmed the presence of exosomal markers (CD9, TSG101 and flotillin-2) in all the exosomal preparations obtained with the different isolation methods (Fig 4B). The NTA analysis (Fig 4C) revealed that the size distribution of the isolated particles was similar for those obtained with ExoQuick (mean: 120±11.4 nm, mode: 88.9±7.8 nm) and qEV2 (mean: 128.9±6.9 nm, mode: 96.5±6.7 nm). Notably, the size distribution profile of particles isolated by ExoEasy was significantly different compared to the other two isolation procedures, with more vesicles with higher size (mean: 174±8.5 nm, mode: 168.8±9.6 nm). We then analyzed the level of VDZ bound to exosomes by ELISA: the amount of VDZ sequestrated in 10¹⁰ exosomes was comparable among the three exosomal preparations (Fig 4D).

Fig 4 — Exosomes were isolated from a pool of patient’s sera by polymer precipitation (Exoquick), membrane-affinity (ExoEasy) and size-exclusion column (qEV2) methods. Vesicles were quantified by Exocet (A), validated for the expression of exosomal markers by immunoblotting (B) and analysed for size distribution by NTA (C). The levels of VDZ bound to exosomes were quantified by ELISA. Data are shown as mean ± SD. * p<0.05 compared to exosomes isolated by ExoEasy and qEV2; § p<0.05 compared to exosomes isolated by ExoQuick and qEV2.

Exosomes compete with T cells for binding to VDZ

To evaluate if VDZ is able to bind exosomes also from individuals never exposed to VDZ, we incubated CTRL exosomes with increasing amount of VDZ and then we analyzed exosomal lysate by native immunoblotting. The results demonstrated that exosomes were able to bind VDZ in a dose–dependent manner, depicting a parabolic curve to saturation (Fig 5A). As negative control, we also incubated VDZ alone with Exoquick in order to exclude its precipitation as a contaminant during the isolation procedure.

Fig 5 — VDZ bound to exosomes (A) or to CD4+ T cells (B) were evaluated by native immunoblotting. One representative blot is shown of three independent experiments. (C) CD4+ T cells were incubated with VDZ with or without exosomes and added to MAdCAM-1-coated plate. Representative microscope images of adherent cells stained with Hoechst dye are shown. Data are shown as mean ± SD. * p<0.05 compared to adherent cells treated with 5 ng/ml VDZ alone.

In order to evaluate if exosomes compete with T cells for VDZ binding, we incubated purified CD4+ T cells with VDZ alone or with a fixed amount of exosomes; the levels of VDZ bound to cells were then measured by native immunoblotting. We found that the amount of VDZ bound to cells was impaired in the presence of exosomes, either if exosomes were pre-incubated with VDZ or given with VDZ simultaneously to CD4+ T cells (Fig 5B). We next evaluated the capacity of exosomes to interfere with the adhesion of VDZ-treated CD4+ T cells (Fig 5C). As expected, VDZ significantly suppressed the adhesion of CD4+ T cells to MAdCAM-1 coated plates. Of note, the number of adherent cells were increased in the presence of exosomes. Hence, the data suggest that exosomes are able to sequestrate VDZ, decreasing the amount available for binding to T cells.

Discussion

Recent studies have revealed that exosome crosstalk mechanisms may influence major IBD-related pathways, such as immune responses, barrier function and intestinal flora [21]. In our study, we propose a completely new role of exosomes in IBDs, whereby vesicles expressing the α₄β₇ integrin might bind VDZ and interfere with drug bioavailability and efficacy.

As with other biologic therapies, a variable proportion of patients do not respond to VDZ at induction or they might lose response over time. Of note, the immunogenicity of VDZ is very low if compared to that of anti-TNF-agents, since the development of anti-VDZ antibodies seems to occur rarely (in 2–4% of cases) [22–24] and therefore does not seem to affect the clinical efficacy of the drug [25]. In fact, anti-VDZ antibodies usually appear after the first infusion, but they are transient [23] and do not correlated with clinical outcomes [22]. In patients who lose response to VDZ over time, the most common management strategy is to reduce the therapeutic interval between VDZ infusions, from 8 to 4 weeks, in order to raise the drug concentration until the next medication dose [26]. Although optimal VDZ trough levels during induction and maintenance have been shown to be associated with endoscopic and clinical outcomes [27], some patients do not respond to this therapy even when adequate trough levels are reached, suggesting that, in these cases, “therapeutic” drug concentrations may be not sufficient to predict clinical response [25]. Therefore, new strategies to predict therapeutic response might be needed.

Our data demonstrate that circulating exosomes express the VDZ target α₄β₇ integrin and bind VDZ, decreasing its bioavailability. Of note, we confirmed the presence of VDZ bound to the vesicles using three different techniques, based on different isolation methods, thus avoiding possible contamination related to a single method.

When VDZ binds to exosomes, it may interfere with their regulatory activity on T cells trafficking into the gut. Exosomes secreted from gut-tropic memory/effector T cells act as a negative regulator that can adjust the levels of gut-specific lymphocyte homing by suppressing MAdCAM-1 expression in the small intestine [28]. Moreover, our data suggest that exosomes could be involved in mediating VDZ resistance by sequestration of therapeutic molecules into vesicles. In accordance with our hypothesis, it has been already reported that exosomes secreted by breast cancer cells carrying HER2 antigen, the target of Trastuzumab, contribute to drug sequestration and compromise its efficacy [18]. Similarly, exosomes released from B-cell lymphomas carrying the CD20 target antigen, may act as decoy targets upon Rituximab exposure, allowing lymphoma cells to escape from humoral immunotherapy [29].

In general, IBD patients previously treated with biologic agents are less likely to respond to a second biologic drug [30]. In our study, we observed that the serum exosomal concentration and the expression of α₄β₇ integrin on vesicles surface was greater in anti-TNFα-antagonist exposed patients compared to anti- TNFα naïve patients. Accordingly, we reported an increased exosomal sequestration of VDZ in anti-TNFα-antagonist exposed patients, suggesting that previous biologic therapy may have altered the sequestration capacity of circulating exosomes, thus reducing the efficacy of VDZ in patients who failed anti-TNFα agents. In agreement, a trend of significant correlation was found between exosomal integrin expression and drug sequestration.

Our results could explain the low and slow response rate of VDZ in patients with a large inflammatory burden whereby the efficacy of the drug could be completely or partially compromised by the increase in α₄β₇ integrin expression in exosomes, which could bind and inactivate the drug. However, the initial combination of VDZ and steroids or calcineurin inhibitors could reduce exosome formation, decrease drug sequestration and increase VDZ efficacy [31, 32].

There are several limitations to the present study. The retrospective design and the very low number of patients involved do not allow us to establish a significative correlation between our findings and the clinical and laboratory behavior of the disease. Moreover, the single blood sample obtained at different time points in individual patients might not entirely reflect the disease behavior and the drug efficacy over time.

In conclusion, this is the first report demonstrating that exosomes bind VDZ and that such binding may interfere with its pharmacokinetics. Additional, larger studies will assess whether VDZ sequestration in exosomes may correlate with clinical and endoscopic response and whether it is possible to interfere with such binding to increase drug bioavailability.

Acknowledgments

We thank Prof. Dario Sorrentino (IBD Center, Division of Gastroenterology, Virginia Tech Carilion School of Medicine, Roanoke, Virginia) or its valuable support in proofreading and editing of this work.

Data Availability

All relevant data are within the manuscript.

Funding Statement

This work was supported by Grant Interreg ARTE (J22F170001005) and partially supported by an unrestricted grant from VivaBioCell S.p.A. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0242342.r001

Decision Letter 0

Aftab A Ansari

10 Sep 2020

PONE-D-20-23481

Circulating exosomes express α4β7 integrin and compete with CD4+ T cells for the binding to Vedolizumab

PLOS ONE

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Reviewer #1: The study reported by Domenis et al reports a proof of the existence of a mechanisms that can consume Vedolizumab in vivo, via shedding of exosomes from T cells. This as pointed out in the manuscript is not unheard of as cells release exosomes bearing some but not all their membrane proteins. Of note the circulating exosomes isolated appear predominantly released from T cells based on CD3 expression. The data goes to great lengths to demonstrate the removal of vedolizumab by exosomes, and its ability to affect T cell binding to MAdCAM-1 in vitro. There is also ex vivo demonstration demonstration of a4B7 expression on plasma exosomes, but the study did not include additional data to show that the therapy failure was actually due to exosomes consuming the mAb. There are several concerns about the manuscript in its current form:

1) Study population: The number of patients is very small and heterogeneous. Individual ages are not listed and that leaves the reader to wonder whether select associations may be possible with exosome recovery etc. There was no scoring of UC included. Since the conclusions suggest that exosomes might have contributed to the failure of the Vedolizumab therapy at the very least, ADA should have been checked against the same Mab. It is quite possible that patients failing anti-TNF-a may have developed ADA that either cross reacts with, or at least cross primes the patient for Vedolizumab. As a more minor point, the text mentions “naïve patients” several times which is confusing. Specify “anti-TNF-a naïve”. Next, the table of patients lists 6 patients in the TNF-a naïve group as having undergone anti-TNF agent therapy??? Which is it?

2) Considering that most exosomes are born from T cells, It would have been quite informative also to have flow cytometry conducted on CD4 and CD8 T cells for activation markers including a4B7 and test whether these expression markers correlate with the level of a4B7 expressed by exosomes.

3) Minor point: the controls are “BD” which is very close to IBD, perhaps an unfortunate choice for the reader.

4) Figure 1A,B: There is quite a bit of variability in the amount of exosomes isolated. Is the isolation method truly quantitative? Controls have higher non significant numbers of exosomes than all 17 UC patients together. But then, the anti-TNF-exposed patient samples are statistically higher though that is driven only by 3 values. This was not really addressed and one wonders how reliable these values might be.

5) Figure 2: Same concern for the a4B7 exposure in samples from only 2 anti-TNF-a exposed patients. This could be due to limited numbers since there is no difference with controls. The same applies to Fig 3C.

6) Figure 3B is confusing as presented. Exosomes from control patient cannot contain VDZ since they did not under such treatment, this needs to be specified in the figure legend.

7) Figure 4A and B raise new questions regarding the various exosome purification methods. There are quantitative but also qualitative differences in the isolation of exosomes that are not commented upon. The qEV2 method, appear far less efficient but then again these exosomes express markedly more Flottilin than others. What does that mean? Different subtypes of exosomes isolated? At the very least these should be characterized for the CD3 and CD14 expression. The scale of Fig 4A needs to be changed to present the actual numbers purified even via the qEV2 method.

8) Figure 5: How many replicates were performed for the binding curve? There were no error bars. The picture in 5C are also very dark and difficult to read. If possible provide lighter ones.

Reviewer #2: In this manuscript Domenis et al., made attempts to characterize exosomes isolated from serum of ulcerative colitis patients treated with VDZ and address the possibility of circulating exosomes bind VDZ and interfere with its therapeutic efficacy.

However, It is well established in the field that EVs coming out of a4b7 expressing cells carry the a4b7 on their surface and it is obvious that after infusion of mAB against a4b7 for treatment of IBD, a fraction of the mab is going to binds to these EVs.

The major question is that, how this EV bound Mabs effects the therapeutic concentration and bioavailability of the mab? And how that phenomenon going to reduce the masking of a4b7 within the a4b7 expressing gut homing lymphocytes and is responsible for therapeutic resistance to VDZ? was not addressed mechanistically.

Although the authors demonstrated that a4b7 expressing EVs bound VDZ in serum, but how that compromise the masking of a4b7 in gut homing lymphocyte remain unanswered?

The most relevant experiment to prove that, binding of VDZ to EVs reduce the effective therapeutic concentration of VDZ and is responsible to VDZ resistance, is to simply show the % masking of a4b7 that is achieved within the a4b7+ gut homing lymphocytes from the clinical sample population used in the study? Therefore, it will great idea that authors can performing this experiment to establish the hypothesis presented in the manuscript that EV bound VDZ based lowering of VDZ bioavailability is responsible for therapeutic resistance of VDZ?' will be great contribution to the VDZ field.

Otherwise hypothesis and idea is good but important experiment is missing?

**********

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PLoS One. 2020 Nov 12;15(11):e0242342. doi: 10.1371/journal.pone.0242342.r002

Author response to Decision Letter 0

28 Oct 2020

Reviewer #1

The study reported by Domenis et al reports a proof of the existence of a mechanisms that can consume Vedolizumab in vivo, via shedding of exosomes from T cells. This as pointed out in the manuscript is not unheard of as cells release exosomes bearing some but not all their membrane proteins. Of note the circulating exosomes isolated appear predominantly released from T cells based on CD3 expression. The data goes to great lengths to demonstrate the removal of vedolizumab by exosomes, and its ability to affect T cell binding to MAdCAM-1 in vitro. There is also ex vivo demonstration of a4B7 expression on plasma exosomes, but the study did not include additional data to show that the therapy failure was actually due to exosomes consuming the mAb. There are several concerns about the manuscript in its current form:

As suggested, demographic and disease characteristics, including Mayo score, of the each patient were listed in two new table, in which patients were subdivided in anti-TNFα naïve and anti-TNFα antagonist exposed.

Since the conclusions suggest that exosomes might have contributed to the failure of the Vedolizumab therapy at the very least, ADA should have been checked against the same Mab. It is quite possible that patients failing anti-TNF-a may have developed ADA that either cross reacts with, or at least cross primes the patient for Vedolizumab.

Anti-TNF therapies and Vedolizumab act against different targets that do not cross-react. There is no evidence that ADA developed against anti-TNF agents may cross-react or prime patients when treated with Vedolizumab. The ELISA kit we used to test ADA against anti-TNF agents are specific for these therapeutic agents and the ELISA we used to test ADA against Vedolizumab are specific for this drugs and gave negative results in patients who previously developed ADA against anti-TNF agents. But, it is well recognized that patients developing ADA against a first biologic agent are at risk to develop an immune response against other biological agents in general. Perhaps, with the sentence “cross prime”, the Reviewer intended that a patients is prone to develop immunereactivity against VDZ, but exosomes eventually implicated in anti-TNF agents sequestration acts differently compared to those binding VDZ since the target on exosomes is different (TNF versus α4β7) and the ADA specifically directed against a different drug.

As a more minor point, the text mentions “naïve patients” several times which is confusing. Specify “anti-TNF-a naïve”.

We specify “anti-TNFα naïve patients” throughout the text, as suggested by reviewer.

Next, the table of patients lists 6 patients in the TNF-a naïve group as having undergone anti-TNF agent therapy??? Which is it?

We apologized for the mistake.

The suggestion of the reviewer is very interesting, but we cannot do this experiment since we used the samples stored for this study to test exosomes and we do not have T cells frozen available for this experiment, but we take into consideration this suggestion for the next studies.

3) Minor point: the controls are “BD” which is very close to IBD, perhaps an unfortunate choice for the reader.

We agree with reviewer and we replace “BD” with “CTRL”.

We agree that the difference between TNF-N and TNF-E in exosomal concentration and integrin expression was near the limit of significance (p=0.047), but was supported by the significant increased sequestration of VDZ in TNF-E compared to TNF-N patients (61.5 ± 26% versus 36.7 ±16.8%; p=0.037). In other words, the higher number of exosomes combined with a higher expression of integrin in TNF-E compare to TNF-N patients results in a greater drug sequestration. Obviously, we believe that exosomes may be only one of the causes of the failure or loss of response to therapy.

We agree with the reviewer that the number of clinical cases is low, but we are already started a second study with a wider patients population aimed to confirm our observations.

6) Figure 3B is confusing as presented. Exosomes from control patient cannot contain VDZ since they did not under such treatment, this needs to be specified in the figure legend.

We thank the reviewer for this observation and we have modified the text specifying that “as expected, exosomes isolated from serum of CTRL patient was negative for VDZ as they are not under therapy, while the signal was present in UC patients.”

Since the discovery of exosomes, a gold standard method for their isolation has not yet been identified and the technique used by different research groups was always a compromise between purity and yield. It is clearly recognized that the different exosomal enrichment methods yield different subpopulations, which could be can be distinguished on the basis of size and expression of the exosomal markers. For example, large EXOs secreted from mesenchymal stem cells were found to be enriched in CD63 and flotillin-1, whereas small EXOs were enriched in ALIX and TSG101 (Willis GR et al. Toward exosome-based therapeutics: isolation, heterogeneity, and fit-for-purpose potency. Front Cardiovasc Med (2017) 4:63.10.3389/fcvm.2017.00063).

For these reason, the International Society for Extracellular Vesicles (ISEV) specifies that there is no single optimal separation method and the choice must be made based on the downstream applications and scientific question. In addition, it recommends to validate data by testing different isolation methods (Lötvall et al. (2014) Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles, Journal of Extracellular Vesicles, 3:1, 26913, DOI: 10.3402/jev.v3.26913).

Accordingly, we did such comparative measures to show that, regardless the isolation technique we used , VDZ binds to the exosomes and the quantity does not change. Consequently, the data reported were not artifacts due to the specific isolation process or the enrichment in a specific exosomal subpopulation.

To better represent the actual number of exosomes obtained by the qEV2 isolation method, we modified the scale of the graph in figure 4A as suggested.

8) Figure 5: How many replicates were performed for the binding curve? There were no error bars. The picture in 5C are also very dark and difficult to read. If possible provide lighter ones.

We specify that one representative blot is shown of three independent experiments in figure legend and we insert the error bars in the graph.

As suggested, we insert lighter and bigger images in picture 5C.

Reviewer #2

In this manuscript Domenis et al., made attempts to characterize exosomes isolated from serum of ulcerative colitis patients treated with VDZ and address the possibility of circulating exosomes bind VDZ and interfere with its therapeutic efficacy.

Although the authors demonstrated that a4b7 expressing EVs bound VDZ in serum, but how that compromise the masking of a4b7 in gut homing lymphocyte remain unanswered?

Otherwise hypothesis and idea is good but important experiment is missing?

We greatly appreciated the suggestion of the reviewer and we believe that the proposed experiment may be useful to further improve our data.

Nevertheless, the aim and originality of this study was to demonstrate that exosomes expressing α4β7 on their surface can bind VDZ and thus potentially interfere with drug availability and possibly, therapeutic efficacy.

To our knowledge, with the exception of the study published by Ciravolo on Trastuzumab, our study is the only one to highlight that exosomes can reduce drug availability. We therefore agree that the number of patients in the present study is low, but sufficient to demonstrate our aim.

We are already working on a much larger series of patients who will be tested either at induction and during the maintenance phases and VDZ sequestration rate by exosomes will be correlated with the clinical response.

Attachment

Submitted filename: Response to reviewers VDZ exo DEF.docx

Click here for additional data file.^{(25.8KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0242342.r003

Decision Letter 1

Aftab A Ansari

2 Nov 2020

Circulating exosomes express α4β7 integrin and compete with CD4+ T cells for the binding to Vedolizumab

PONE-D-20-23481R1

Dear Dr. Fabris,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements and you have added the sentence I have recommended within the text, if this sentence is accurate.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

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Kind regards,

Aftab A. Ansari, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

I would suggest that you add a sentence at the end of lines 161-162 such as "Monitoring of ADA against TNF-alpha and VDZ was performed utilizing non-cross reactive ADA detection ELISA kits from Progenica Biopharma-Grisfol according to the manufacturer's instruction. None of the sera showed detectable levels of ADA.

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0242342.r004

Acceptance letter

Aftab A Ansari

4 Nov 2020

PONE-D-20-23481R1

Circulating exosomes express α₄β₇ integrin and compete with CD4+ T cells for the binding to Vedolizumab

Dear Dr. Fabris:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Aftab A. Ansari

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Attachment

Submitted filename: Response to reviewers VDZ exo DEF.docx

Click here for additional data file.^{(25.8KB, docx)}

Data Availability Statement

All relevant data are within the manuscript.

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PERMALINK

Circulating exosomes express α4β7 integrin and compete with CD4+ T cells for the binding to Vedolizumab

Rossana Domenis

Marco Marino

Adriana Cifù

Giulia Scardino

Francesco Curcio

Martina Fabris

Roles

Abstract

Introduction

Methods

Study population

Table 1. Baseline characteristics of the IBD patients anti TNF-α failure.

Table 2. Baseline characteristics of the IBD patients naïve to TNF-α.

Exosome isolation

Exosome characterization

Analysis of VDZ binding to exosomes

Analysis of exosome competition for binding to VDZ

MAdCAM adhesion assay

Statistical analysis

Results

Purification and characterization of serum exosomes from patients and controls

Fig 1. Circulating exosomes isolation and characterization.

Serum-derived exosomes expressed the α4β7 integrin and were able to bound VDZ

Fig 2. Circulating exosomes express α4β7 integrin.

Fig 3. VDZ is bound to exosomes in UC patients.

Fig 4. Comparison of three different methods of exosome isolation.

Exosomes compete with T cells for binding to VDZ

Fig 5. Exosomes compete with T cells for binding to VDZ.

Discussion

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Aftab A Ansari

Roles

Author response to Decision Letter 0

Decision Letter 1

Aftab A Ansari

Roles

Acceptance letter

Aftab A Ansari

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Circulating exosomes express α₄β₇ integrin and compete with CD4+ T cells for the binding to Vedolizumab

Serum-derived exosomes expressed the α₄β₇ integrin and were able to bound VDZ

Fig 2. Circulating exosomes express α₄β₇ integrin.