ORIGINAL RESEARCH published: 16 December 2021 doi: 10.3389/fmed.2021.781239 B Cell-Derived Extracellular Vesicles Reveal Residual B Cell Activity in Kidney Graft Recipients Undergoing Pre-Transplant Desensitization David Cucchiari 1,2*, Valeria Tubita 2 , Jordi Rovira 2,3 , Maria J. Ramirez-Bajo 2,3 , Elisenda Banon-Maneus 2,3 , Marta Lazo-Rodriguez 2 , Natalia Hierro-Garcia 2 , Francesc E. Borràs 3,4,5 , Pedro Ventura-Aguiar 1 , Gastón J. Piñeiro 1 , Jaume Martorell 6 , Lluís Peri 7 , Mireia Musquera 7 , Alexandre Hertig 8 , Federico Oppenheimer 1,3 , Josep M. Campistol 1,2,3 , Fritz Diekmann 1,2,3 and Ignacio Revuelta 1,2,3* 1 Department of Nephrology and Kidney Transplantation, Hospital Clínic, Barcelona, Spain, 2 Laboratori Experimental de Edited by: Nefrologia i Trasplantament (LENIT), Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain, Kathrin Eller, 3 Red de Investigación Renal (REDINREN), Madrid, Spain, 4 REMAR-IVECAT Group, “Germans Trias i Pujol” Health Science Medical University of Graz, Austria Research Institute, Badalona, Spain, 5 Department of Cell Biology, Physiology and Immunology, Universitat Autònoma de Reviewed by: Barcelona, Cerdanyola del Vallès, Spain, 6 Department of Immunology, Hospital Clínic, Barcelona, Spain, 7 Department of Miha Arnol, Urology, Hospital Clínic, Barcelona, Spain, 8 Service de Néphrologie, Hôpital Foch, Suresnes, France University Medical Centre Ljubljana, Slovenia Konstantin Doberer, Background: Living-donor kidney transplant (LDKT) recipients undergoing Medical University of Vienna, Austria desensitization for Human Leukocyte Antigen (HLA)-incompatibility have a high *Correspondence: risk of developing antibody-mediated rejection (ABMR). The purpose of the study is Ignacio Revuelta irevuelt@clinic.cat to evaluate if residual B cell activity after desensitization could be estimated by the David Cucchiari presence of circulating B cell-derived extracellular vesicles (BEVs). cucchiari@clinic.cat Methods: BEVs were isolated by Sepharose-based size exclusion chromatography Specialty section: and defined as CD19+ and HLA-II+ extracellular vesicles. We analyzed stored serum This article was submitted to Nephrology, samples from positive crossmatch LDKT recipients before and after desensitization at a section of the journal first post-transplant biopsy and at 12-month protocol biopsy (n = 11). Control groups Frontiers in Medicine were formed by hypersensitized patients who were not submitted to desensitization Received: 22 September 2021 (n = 10) and by low-risk recipients (n = 9). A prospective validation cohort of 11 Accepted: 15 November 2021 Published: 16 December 2021 patients also included the analysis of B cells subpopulations in recipients’ blood and Citation: lymph nodes recovered upon graft implantation, along with BEVs analysis before and Cucchiari D, Tubita V, Rovira J, after desensitization. Ramirez-Bajo MJ, Banon-Maneus E, Lazo-Rodriguez M, Hierro-Garcia N, Results: We found out that CD19+ and HLA-II+BEVs dropped significantly Borràs FE, Ventura-Aguiar P, after desensitization and relapse in patients who later developed ABMR was Piñeiro GJ, Martorell J, Peri L, Musquera M, Hertig A, evident. We validated these findings in a proof-of-concept prospective cohort Oppenheimer F, Campistol JM, of 6 patients who received the same desensitization protocol and also in a Diekmann F and Revuelta I (2021) B control group of 5 LDKT recipients. In these patients, B cell subpopulations Cell-Derived Extracellular Vesicles Reveal Residual B Cell Activity in were also studied in recipients’ blood and lymph nodes that were recovered Kidney Graft Recipients Undergoing before the graft implantation. We confirmed the significant drop in BEVs after Pre-Transplant Desensitization. Front. Med. 8:781239. desensitization and that this paralleled the reduction in CD19+cells in lymph doi: 10.3389/fmed.2021.781239 nodes, while in peripheral blood B cells, this change was almost undetectable. Frontiers in Medicine | www.frontiersin.org 1 December 2021 | Volume 8 | Article 781239 Cucchiari et al. B cell Extracellular Vesicles in Kidney Transplantation Conclusions: BEVs reflected B cell residual activity after desensitization and this could be a valid surrogate of humoral alloreactivity in this setting. Keywords: B cells, kidney transplantation, desensitization, HLA-incompatibility, extracellular vesicles (EV), exosomes, plasma cells INTRODUCTION importance of transplant immunosuppression in EV content from colorectal cancer cell lines in the regulation of the pre- Patients with chronic kidney disease that are sensitized to metastatic niche (16). It is reasonable to speculate that circulating HLA antigens have limited access to kidney transplantation, BEVs reflect B cell proliferation in bone marrow and lymphoid resulting to increased mortality in the waiting list. A possibility organs, even though the circulating B cells are not detectable to overcome the HLA barrier is represented by desensitization because of desensitization. To prove this hypothesis, we before kidney transplantation. This regimen, which is usually developed a retrospective and a prospective study in living-donor based on plasma exchange, intravenous immunoglobulins, and kidney transplant (LDKT) recipients who were undergoing a anti-CD20 antibodies, is being done in order to reduce the desensitization protocol based on anti-CD20 antibodies, plasma quantity of circulating donor-specific antibodies (DSAs) and to exchanges, and intravenous immunoglobulins. deplete B cells (1–3). This option is associated with favorable results, as graft and patient survival are undoubtedly better when MATERIALS AND METHODS compared to hypersensitized patients on dialysis waiting for a compatible donor (4). However, despite the reasonable solid Study Population outcomes, incidence of antibody-mediated rejection (ABMR) is The retrospective cohort included patients who had kidney as high as 30–50% (5–7), and forces a transplant physician to transplantation procedure across a 10-year period (2006– enhance immunosuppression but with concerns of higher rate of 2015). Demographics and clinical characteristics of both donors infectious, neoplastic and cardiovascular complications (7, 8). and recipients have been collected, along with immunological The effects of desensitization on DSA titer can be controlled profiles of recipients, HLA-matching, and immunosuppressive using solid-phase techniques. However, how B cell biology is treatment. Patients were divided into three groups according to affected by this treatment remains unclear. One possibility is the immunological risk: patients who received desensitization to think that circulating B cells represented a good estimate before transplantation for a positive crossmatch (“DS” group, n of treatment efficacy. However, while circulating B cells are = 11), hypersensitized patients with a cPRA I+II > 85% not easily depleted after a single dose of anti-CD20 antibody, a high submitted to desensitization and without a DSA (“HS” group, proportion of B cells survived in lymph nodes with a switched- n = 10), and a low-risk group with baseline cPRA I+II < 10% memory phenotype (9). To check activity and proliferation of and no DSAs (“CT” group, n = 9). For those patients who were residual B cells after desensitization, a fine-needle aspiration of transplanted before 2008, the year in which Luminex study was bone marrow or lymph node biopsy is unpractical, apart from routinely implemented in the transplant work-up, patients were being invasive. Therefore, we hypothesized that the activity of B assigned to the HS group if they had been re-transplanted and if cells, which survived in lymphoid organs after desensitization, they had lost their previous graft for rejection (two cases). can be estimated by the presence of circulating B cell-derived Induction was based on anti-thymocyte globulins, extracellular vesicles (BEVs). monoclonal anti-CD25 antibodies, or omitted according to Extracellular Vesicles (EVs) are a heterogeneous population the immunological risk. Maintenance was based on tacrolimus, of cell-derived vesicles of various origins and sizes, including mycophenolate, and steroids. Desensitization was based on exosomes, microvesicles, and apoptotic bodies (10). They played anti-CD20 monoclonal antibodies (Rituximab, Mabthera, Roche, a key role in intercellular communication, delivering signal Basel, Switzerlad, 1 or 2 doses of 400 mg) and on plasma molecules (proteins, nucleic acids, lipids, etc.) that can regulate exchanges (total number according to DSA titer or repeated XM immune functions (11). Concretely, EVs derived from immune measurements), followed by intravenous immunoglobulins every cells are implicated in antigen presentation, immunoregulation, two exchanges (Plangamma, 200 mg/Kg for session, Institute and viral transmission (12, 13). B cells actively secrete EVs upon Grifols, Barcelona, Spain). proliferation stimuli, such as T-cell help via CD40 and IL-4 The three time-points examined in the retrospective study signaling (11, 13). Importantly, BEVs display markers of B cell were as follows: (i) transplantation day; (ii) first biopsy (CD19, IgM, IgG) and of antigen-presenting cells origin (HLA-I, (either for indication or for-protocol); and (iii) one-year after HLA-II, CD86) (11). transplantation. In our institution, kidney biopsies are performed The possibility of circulating EVs to act as efficient biomarkers per-protocol at 3 and 12-months after kidney transplantation. has been recently highlighted in different fields. In oncology, for Regarding the second time-point, we examined the sample of example, tumor-derived circulating exosomes are associated with the first per-indication biopsy in those cases in which rejection the burden of the primary mass (14). In kidney transplantation, developed before the 3-month per-protocol biopsy. In all the mRNA transcripts from circulating exosomes are associated with other cases, the 3-month per-protocol biopsy sample was studied. rejection phenotypes (15). Our group recently highlighted the This choice was made in order to avoid the potential effects Frontiers in Medicine | www.frontiersin.org 2 December 2021 | Volume 8 | Article 781239 Cucchiari et al. B cell Extracellular Vesicles in Kidney Transplantation of B cell depleting treatment on BEVs content of the second equipped with a 638 nm laser and CCD camera (model F-033). time-point. Pathological examination and diagnosis of ABMR Data were analyzed with the Nanosight NTA Software version 3.1 were updated according to Banff 2017 criteria (17). Treatment (build 3.1.46). Representative samples were evaluated in sterile- of ABMR was based on anti-CD20 monoclonal antibodies filtered PBS 1X (n = 3). Readings were taken in single capture or (Rituximab, Mabthera, Roche, Basel, Switzerlad, 1 dose of triplicates for 60 s at 30 frames per second, and through manual 400 mg at the beginning and 1 dose at the end of the cycle monitoring of temperature (Supplementary Figure 2). of 400 mg) and 6 plasma exchanges, followed by intravenous immunoglobulins every two exchanges (Plangamma, 200 mg/kg Complement-Dependent Cytotoxicity and for session, Institute Grifols, Barcelona, Spain). Regarding the prospective cohort, all patients were Flow Cytometry Cross-Match transplanted from 2018 to 2019, with the same desensitization, Complement-Dependent Cytotoxicity (CDC) crossmatch was induction, and maintenance protocol employed as in the performed with peripheral blood mononuclear cells (PBMCs) retrospective cohort. of the donor, according to the NIH Technique. To rule out the Extracellular vesicles were studied according to the MISEV presence of autoantibodies, all crossmatch tests were carried out guidelines of 2018 (18). A checklist with all the items can be with sera, with and without dithiothreitol treatment, in order retrieved in Supplementary Material. to discard IgM. The T and B cell flow cytometry crossmatch was performed using freshly obtained PBMCs from the donor as Isolation of EVs by Sepharose-Based well. The T and B cells were identified using mouse anti-human CD19 and CD3 antibodies. Goat antihuman F(ab’)2 IgG(γ) was Size-Exclusion Chromatography used to identify anti-HLA IgG attached to the cells (Clones Stored serum samples at −80◦ C were thawed in ice. After two indicated in Supplementary Table 2). The shift of the median centrifugation steps at 2000 g, to eliminate cellular debris,1 ml channel fluorescence (SMCF) between the test sera and the of the serum was loaded on a Sepharose column, as previously negative control sera was used to assign positivity or negativity. described (19, 20). Concisely 10 ml syringes (Becton Dickinson; The cut-off values were assigned according to the median + 3 San Jose, United States) were stacked with 10 ml of Sepharose CL- standard deviations of 20 non transfused male Single Antigen test 2B (GE Healthcare; Uppsala, Sweden). Sepharose was previously negative sera. washed with 0.32% sodium citrate PBS 0.22 µm-filtered. The tip of the syringe was stuffed with nylon stockings (40 denier). Immediately after sample load, it was followed by elution with Assessment of DSAs and Calculated PRA 0.32% sodium citrate PBS 0.22µm-filtered and 18 fractions of by Luminex Technology 0.5 ml volume were sequentially collected. These fractions were Anti-HLA antibodies were assessed before transplantation and analyzed through a spectrophotometer at 280 nm absorbance during the per-protocol and the for-cause renal biopsy through to estimate protein contents (NanodropTM, ThermoFisher; Luminex-based technology (22). Briefly, antibodies were tested Waltham, MA, USA). using Single Antigen Bead test (LIFECODES R Single Antigen, Immucor, Georgia, USA). An allele was considered positive if Characterization of EVs by Flow Cytometry the MFI was over 1,500 and was x4 times higher than the The following step was the coupling of fraction 6–13 (50 µl for Lowest Reactive Antigen (LRA) of the same locus. Calculated sample) to 5 µl of latex beads (4 µm in diameter) for 15 min PRA (cPRA) was determined using a panel of 500 unrelated and then with 1 ml of BCB buffer (PBS 0.1% BSA). After 1 night individuals from local population and was typed for A, B, C, rotation at room temperature, fractions were incubated in a 96- DRB1, and DQB1 in high resolution (2 fields) by sequence- well plate with two primary antibodies against the EV-specific based typing. tetraspanins biomarkers CD9 and CD81 (final dilution 1:10) (20). After careful washing and decanting, wells were charged with 200 µl of PBS 0.22µm-filtered before flow-cytometry study with Flow Cytometry Analysis of PBMC and the FACS-Fortessa cytometer (BD Biosciences; San Jose, CA, Lymph Nodes United States) was performed. For every well, 10.000 beads for Peripheral blood mononuclear cells (PBMCs) were isolated sample were examined and MFI was used to calculate the content from patient blood samples by centrifugation on a density of EVs in the different fractions (Supplementary Figure 1). gradient (400 g for 30 min in Ficoll-paque PREMIUM, GE Those fractions that proved to be enriched in EVs by high Healthcare, Madrid, Spain). Lymph nodes were mashed and expression of CD9 and CD81 were later pooled and analyzed passed through a 70 µm nylon cell strainer (BD Falcon) and for markers of B cells (CD19 and HLA-II) (21). The MFI value single-cell suspensions were obtained. Cell surface markers were for CD19 and HLA-II was normalized to the CD9 biomarker of stained with antibodies indicated in Supplementary Table 3, exosomes analyzed in the same pool. All antibodies for exosome according to the instructions of the manufacturer. In all samples, characterization are indicated in Supplementary Table 1. Aqua Live/Dead fixable dead cell kit (Thermo Fisher Scientific, Waltham, MA, USA) was used unambiguously to remove dead Nanoparticle Tracking Analysis (NTA) cells. Flow cytometry analysis was performed on a FACS Canto Size distribution and concentration of EVs were measured using II (BD Biosciences, Heidelberg, Germany). Data were analyzed the NanoSight LM10 instrument (Malvern, United Kingdom), using FlowJo software (Tree Star, Ashland, OR, USA). Frontiers in Medicine | www.frontiersin.org 3 December 2021 | Volume 8 | Article 781239 Cucchiari et al. B cell Extracellular Vesicles in Kidney Transplantation Transmission Electron Microscopy (TEM) TABLE 1 | Baseline characteristics (above) and B cell-derived extracellular A Holey Carbon support film on a 400-mesh copper grid was vesicles (BEVs) assessment (below) in the three study groups. used. After the glow discharge, the sample was deposited onto Control (CT) Hypersensitized Desensitized the grid, which was mounted on a plunger (Leica EM-CPC) n=9 (HS) n = 10 (DS) n = 11 and blotted with Whatman No. 1 filter paper. The suspension was vitrified by rapid immersion in liquid ethane. The grid was Age (years) 41.9 ± 9.94 43.2 ± 6.86 45.4 ± 15.64 mounted on a Gatan 626 cryo-transfer system and was inserted Sex (%males) 3/9 (33.3%) 4/10 (40.0%) 5/11 (45.5%) into the microscope. Images were obtained using a Jeol JEM Dialysis vintage (months) 20.8 ± 22.3 65.8 ± 52.3 49.3 ± 78.1 2011 cryo-electron microscope operated at 200 kV, recorded on a Hypertension (% yes) 8/9 (88.8%) 8/10 (80.0%) 9/11 (81.8%) Gatan Ultrascan US1000 CCD camera, and were analyzed with a Diabetes mellitus (% yes) 2/9 (22.2%) 1/10 (10.0%) 3/11 (27.3%) Digital Micrograph 1.8 (n = 3 per group). Previously transplanted 2/9 (22.2%) 5/10 (50.0%) 6/11 (54.5%) (% yes) Statistical Analysis Donor age (years) 52.0 ± 14.2 55.3 ± 13.3 54.4 ± 10.3 Data are presented as number and/or percentages, mean Donor sex (%males) 5/9 (55.5%) 4/10 (40.0%) 4/11 (36.4%) and standard deviation, or median and interquartile range, HLA A-B-DR incompatibilities 3.11 ± 1.90 2.20 ± 2.46 3.91 ± 1.57 as indicated. Statistical comparison among groups has been cPRA I+II at baseline (%) 0 95.6 ± 3.8 78.4 ± 21.4 performed through Student’s t-test, Mann-Whitney, or one-way Induction (%) ANOVA with LSD post-hoc analysis, as appropriate depending None 1/9 (11.1%) / / on data distribution and number of groups. Correlation between Anti-CD25 antibodies 5/9 (55.5%) 1/10 (10.0%) / continuous variables has been explored with Pearson’s analysis. Lymphocyte-depleting 3/9 (33.3%) 9/10(90.0%) 11/11 (100.0%) Time-dependent association with rejection of the biomarkers in agents the retrospective analysis has been analyzed with mixed model Creatinine +3 months (mg/dl) 1.22 ± 0.48 1.59 ± 0.49 1.55 ± 0.60 linear rejection. All statistical tests have been conducted with a Creatinine +12 months 1.23 ± 0.53 1.65 ± 0.58 1.69 ± 0.58 95% confidence interval, and a p < 0.05 has been considered (mg/dl) significant and has been highlighted with an asterisk in the Desensitization parameters Figures. To carry out all the above-mentioned analysis, software DSA (MFI) / / 6,130 ± 3,405 SPSS v.20 (SPSS Inc., Chicago, IL, USA) and GraphPad v.5 Rituximab (mg) / / 618.2 ± 208.9 (GraphPad Software, La Jolla, CA, USA) have been used. The Plasma exchanges (n) / / 5 [2–7] local Ethical Committee approved the study. HLA, Human Leukocyte Antigen; cPRA, calculated Panel-Reactive Antibodies. RESULTS Baseline Characteristics of the Studied based on tacrolimus, mycophenolate, and prednisone in all groups (Table 1). Population B cell-derived extracellular vesicles (BEVs) were extracted from stored serum samples of 30 LDKT recipients in the following Circulating BEVs Can Be Detected During time-points: transplantation day, first biopsy (either per-protocol the First Year After Kidney Transplantation or per-cause), and 12-month protocol biopsy. This population Isolation and characterization of microparticles were included the study group of 11 desensitized patients for a positive consistent with EV surface biomarkers (CD9 and cross-match (DS group), while control groups were represented CD81) and with the diameter according to flow- by 10 hypersensitized patients (defined as baseline cPRA I+II > cytometry (Supplementary Figure 1), NTA analysis 85%) not submitted to desensitization (HS group), and nine low- (Supplementary Figure 2) and electronic Microscopy risk patients (defined as baseline cPRA I+II < 10%, CT group). (Supplementary Figure 3). BEVs were defined as either In the DS group, before starting the desensitization protocol, a CD19+ or HLA-II+ EVs and were detected all along the first stored serum sample was also available and studied in seven of the year after the kidney transplantation in the three groups (CT, HS, 11 patients. One patient of the DS group died of metastatic colon and DS) (Figure 1), without significant difference at ANOVA cancer before completing the 12-month follow-up. Baseline analysis (Table 2). However, when comparing the DS group with characteristics revealed no differences among groups in terms the similar risk controls (HS group), there was a tendency toward of age, sex, diabetes, hypertension, and donor characteristics. lower CD19+ EVs at transplantation (p = 0.068, Figure 1A), at Calculated PRA (cPRA) I+II at baseline was higher in the first biopsy (p = 0.101, Figure 1B), and lower HLA-II+ EVs at HS and DS group as expected. Causes for desensitization in first biopsy (p = 0.053, Figure 1E). the DS group were a positive flow-cytometry crossmatch in 8 patients and DSA in 3 patients. Of the 11 patients, 10 had a BEVs Drop After Desensitization and detectable DSA by Luminex before transplantation (mean MFI Relapse During ABMR 6,130 ± 3,405) and the only patient without a DSA had a positive Antibody-mediated rejection (ABMR) during the first year after flow-cytometry crossmatch for both T and B cells. Induction kidney transplantation occurred in seven of the 11 patients was most frequently based on lymphocyte-depleting agents in of the DS group. Of the seven rejections in the DS group, the HS and DS group, while baseline immunosuppression was four were discovered at 3-month protocol biopsy, and three Frontiers in Medicine | www.frontiersin.org 4 December 2021 | Volume 8 | Article 781239 Cucchiari et al. B cell Extracellular Vesicles in Kidney Transplantation FIGURE 1 | B cell-derived Extracellular Vesicles (BEVs) evolution along the first year after kidney transplantation at the time of Transplantation (A,D), first biopsy (B,E) and 12-month protocol biopsy (C,F). BEVs (CD19+ and HLA-II+ EVs) were analyzed in the study groups in the three time-points. transplantation day, first biopsy, and 12-month protocol biopsy (ANOVA and LSD Post-hoc analysis). TABLE 2 | BEVs assessment (below) in the three study groups at transplantation, first renal biopsy, and at 12-month protocol biopsy. Control (CT) n = 9 Hypersensitized (HS) n = 10 Desensitized (DS) n = 11 P-value (ANOVA) Transplantation (BEVs) CD19+ EVs 0.97 ± 0.77 1.50 ± 1.04 0.84 ± 0.44 0.148 HLA-II+ EVs 0.66 ± 0.20 0.73 ± 0.31 0.62 ± 0.27 0.639 First renal biopsy (BEVs) CD19+ EVs 0.96 ± 0.47 1.46 ± 0.91 0.88 ± 0.40 0.102 HLA-II+ EVs 0.71 ± 0.24 0.85 ± 0.15 0.64 ± 0.22 0.097 12-month renal biopsy (BEVs) CD19+ EVs 0.91 ± 0.39 1.29 ± 0.50 0.99 ± 0.40 0.337 HLA-II+ EVs 0.72 ± 0.21 0.83 ± 0.20 0.71 ± 0.17 0.403 were discovered before in a per-cause renal biopsy. Within treated except the patient with neoplasia. Only one patient this group, DSAs were present in two of seven patients experienced full recovery after treatment, while the other patients with ABMR (MFI of 3,654 and 4,077, respectively) and in progressed to chronic ABMR at 12-month protocol biopsy. two of four patients without ABMR (MFI of 9,066 and Evolution of CD19+ EVs after transplantation proved to be 6,613, respectively). significantly associated with rejection (p = 0.002), while DSAs We observed a significant drop in both CD19+ and HLA- were not (p = 0.186), according to mixed-model linear rejection II+ EVs after desensitization (CD19+ EVs 1.27 ± 0.30 before analysis (Figures 2E,F). and 0.59 ± 0.25 after desensitization, p = 0.003, and HLA-II+ EVs 0.75 ± 0.06 before and 0.47 ± 0.22 after desensitization, Survival of CD19+CD20- B Cells in Lymph p = 0.024) (Figures 2A,B). Within the DS group, patients Nodes After Desensitization with active ABMR had higher CD19+ and HLA-II+ EVs We developed a prospective validation study to assess the B cell both at first biopsy (n = 7) and at 12 months (n = 5) subpopulations from PBMC and the external iliac lymph node (Table 3 and Figures 2C,D). All the cases of ABMR were recovered upon surgery, along with the circulating BEVs. In this Frontiers in Medicine | www.frontiersin.org 5 December 2021 | Volume 8 | Article 781239 Cucchiari et al. B cell Extracellular Vesicles in Kidney Transplantation FIGURE 2 | Analysis of BEVs in patients undergoing desensitization and their relationship with antibody-mediated rejection (ABMR)–BEVs expressed as CD19+ and HLA-II+ exosomes before and after desensitization in the DS group [(A,B) paired samples t-test] and according to whether or not patients developed ABMR at first biopsy or at 12 months after kidney transplantation [(C,D) Student’s t-test, Table 3]. Evolution of patients according to whether they have rejected (red line) or not (blue line) during the first year is highlighted for MFI of the DSA (E) and CD19+ BEVs (F). Among patients with ABMR, the only patient who did not progress to chronic ABMR and had a normal kidney at following biopsies is highlighted with a reverse triangle. cohort, we studied 11 patients of which six were submitted to The CD3-CD19+ B cells were almost depleted after the same desensitization protocol as above, while five low-risk desensitization in blood (Figure 3C, p = 0.024), but they recipients represented the control group. Baseline characteristics were still present in the lymph node, without statistical of this prospective population are described in Table 4, without difference compared to the control group at the time of any significant difference between the two groups. transplantation (Figure 3D, p = 0.762, and representative There was a total depletion of CD3-CD20+ B cells sample in Figure 4A). On the other side, CD19+CD20+ B from both periphery and lymph node after desensitization. cells were also undetectable in lymph nodes in the DS group, Frontiers in Medicine | www.frontiersin.org 6 December 2021 | Volume 8 | Article 781239 Cucchiari et al. B cell Extracellular Vesicles in Kidney Transplantation TABLE 3 | BEVs evolution in patients submitted to desensitization according to More precise phenotype of B cells revealed that after the clinical outcome. desensitization, there was a loss of naïve B cells (CD27-IgD+) in n CD19+ EVs P-value HLA-II+ EVs P-value lymph nodes, while a substantial proportion of switched-memory B cells (CD27+IgD-) was still alive (Figure 5). First biopsy No rejection 4 0.59 ± 0.40 0.036 0.45 ± 0.23 0.014 ABMR 7 1.13 ± 0.31 0.78 ± 0.13 DISCUSSION 12-month biopsy No rejection 5 0.71 ± 0.18 0.021 0.58 ± 0.10 0.008 Desensitization before LDKT represents a reasonable option ABMR 5* 1.27 ± 0.38 0.84 ± 0.13 for highly sensitized patients in terms of graft and patient survival (4). However, ABMR incidence can be as high as *1missing value (patient death). 30–50% (5–7). This suggests that the biomarkers currently ABMR, antibody-mediated rejection. used to check proper desensitization (DSAs, circulating B cells, flow-cytometry crossmatch) only partially reflect the TABLE 4 | Baseline characteristics of the prospective validation study cohort. complex biology of humoral alloimmunity. In order to check B cell proliferation in secondary lymphoid organs and bone Desensitized Control P-value marrow after desensitization, we propose the use of B n=6 n=5 cells-derived EVs. Age (years) 51.2 ± 12.7 45.2 ± 20.0 0.562 B cell-derived extracellular vesicles (BEVs) are generated Sex (% males) 6 (100.0%) 4 (80.0%) 0.455 by B cells upon differentiation and proliferation stimuli Dialysis vintage (months) 0 [0–48] 8 [3–17] 0.840 (13) and could be a major source of EVs in vivo (11). Hypertension (% yes) 5 (83.3%) 5 (100.0%) 1 In large B cell lymphomas, exosome-derived miRNAs are Diabetes mellitus (% yes) 1 (16.7%) 0 (0.0%) 1 associated with chemotherapy resistance (17). The value Previously transplanted (% yes) 1 (16.7%) 1 (20.0%) 1 of circulating EVs as diagnostic tool has been highlighted Donor age (years) 54.7 ± 4.96 56.6 ± 4.21 0.510 in oncology (14) and in kidney transplantation (15, 16). Donor sex (% males) 0 (0.0%) 0 (0.0%) 1 A recent report also found out that urinary CD3+ EVs HLA A-B-DR incompatibilities 4.00 ± 2.44 3.00 ± 2.23 0.501 are associated with cellular rejection (23). To date, there cPRA I+II at baseline (%) 0 [0–100] 0 [0–46] 0.714 are no studies that specifically assessed BEVs in kidney Creatinine +3 months (mg/dl) 1.49 ± 0.48 1.52 ± 0.37 0.926 transplant recipients. In our experience, we observed the presence of circulating Creatinine +6 months (mg/dl) 1.44 ± 0.28 1.77 ± 0.59 0.420 BEVs in normal and hypersensitized kidney transplant recipients, Desensitization parameters before and within the first year after transplantation, with a non- Isoaglutinins IgG 4 [2–32] significant trend toward higher concentration of BEVs in the Isoaglutinins IgM 4 [2–64] hypersensitized group (Table 2 and Figure 1). Rituximab (mg) 1,000.0 ± 903.3 In patients submitted to pre-transplant desensitization, we Plasma exchanges (n) 4 [1–7] observed a significant reduction in BEVs upon treatment completion (Figures 2A,B) with a significant rebound in patients The desensitized group also included ABO-incompatible recipients, so data about pre- who later developed ABMR (Table 3 and Figures 2C,D). In transplant isoaglutinins IgG and IgM had been added. this prospective study, we observed a complete depletion HLA, Human Leukocyte Antigen; cPRA, calculated Panel-Reactive Antibodies. of CD3-CD20+ both in periphery and in the lymph node. B cells were also assessed as CD3-CD19+ and we also observed that these were almost completely cleared from while a substantial presence of CD19+CD20- B cells still circulation (Figure 4A). In lymph nodes, however, we observed persisted (Figure 4B). a switch from CD19+CD20+ to CD19+CD20- phenotype in desensitized patients (Figure 4B), with a switched-memory Circulating BEVs Correlate With Residual phenotype (Figure 5) and a statistical association with circulating CD3-CD19+ B Cells in Lymph Nodes That CD19+ EVs. Differences, albeit moderate, were statistically Display a Switched-Memory Phenotype significant and also showed that the evolution of CD19+ EVs was We confirmed the significant drop in BEVs after desensitization, associated with the development of rejection while the DSAs were being CD19+ EVs 1.04 ± 0.29 before and 0.80 ± 0.11 after (p not (Figures 2E,F). = 0.024) and HLA-II+ EVs 0.86 ± 0.28 before and 0.73 ± 0.11 Taken together, these findings confirmed that a desensitization after desensitization (p = 0.024), respectively (Figures 3A,B). protocol based on anti-CD20 antibodies, intravenous There was a significant correlation between CD19+ EVs and immunoglobulins, and plasma exchanges is associated with the percentage of CD3-CD19+ B cells in the lymph node (r residual alloimmunity that cannot be detected with the = 0.839, p = 0.037) at the moment of transplantation, while currently available clinical tools. As we observed a significant there was no correlation between circulating CD19+ PBMCs and increase in circulating BEVs in patients experiencing CD3-CD19+ B cells in the lymph node (r = −0.197, p = 0.708). ABMR, we propose that this reflects proliferation and Frontiers in Medicine | www.frontiersin.org 7 December 2021 | Volume 8 | Article 781239 Cucchiari et al. B cell Extracellular Vesicles in Kidney Transplantation FIGURE 3 | Analysis of BEVs and B cells from peripheral blood mononuclear cells (PBMCs) and lymph node in the prospective validation cohort–CD19+ and HLA-II+ BEVs before and after desensitization [(A,B) paired-samples t-test]. At baseline, there is no difference in percentage of circulating B cells (CD3− CD19+ ) between the two groups of patients. Upon desensitization, there is a depletion in circulating CD3− CD19+ cells [(C) Student’s t-test] but a relevant proportion of them persisted alive in the lymph node (D). differentiation of alloreactive B cells resident in secondary lose CD19 expression along their differentiation, CD19+ EVs lymphoid organs and bone marrow that would not be may not reflect the activity of plasma cells, which are ultimately detectable otherwise. responsible for DSA production. However, it seems that non- However, some points need to be addressed before drawing myelomatous plasma cells still express CD19, even though firm conclusions, given the small sample size analyzed in this in a heterogeneous way between individuals (24, 25), while preliminary experience and the generating-hypothesis nature of there is more information about EVs secretion by myelomatous this work. First, while in the DS group, there was a striking plasma cells (26, 27). difference in BEVs before and after desensitization and between In conclusion, in patients undergoing desensitization patients who rejected or not, we did not observe significant before kidney transplantation, we observed a significant differences between the DS and the other groups (Figure 2). decrease in circulating BEVs after treatment. These were This may suggest that the relative changes in BEVs can be associated with the presence of surviving B cells in the lymph meaningful in patients treated with B cell targeted therapies, nodes. Patients who developed ABMR have experienced but at present, this cannot be applied to other population who a significant rebound in circulating BEVs, suggesting receive standard-of-care induction. Second, the two biomarkers proliferation and differentiation of not-circulating alloreactive that we used to define BEVs (CD19 and HLA-II) were not B cells. The next steps will be to analyze BEVs kinetic and specific of only a cell population. The CD19 is expressed function not only in desensitized patients but also in low by B cells in most of their differentiation steps and HLA- and high-risk kidney transplant recipients. Circulating II is also expressed by other antigen-presenting cells such as BEVs may demonstrate to reveal a part of the humoral monocytes-macrophages and dendritic cells. Third, as B cells response that cannot be assessed yet in clinical practice, Frontiers in Medicine | www.frontiersin.org 8 December 2021 | Volume 8 | Article 781239 Cucchiari et al. B cell Extracellular Vesicles in Kidney Transplantation FIGURE 4 | Flow cytometry analysis of circulating and lymph node B cells–(A) After desensitization, CD3− CD19+ B cells disappear from blood circulation (red circle, blue arrow). However, in lymph nodes at the moment of transplantation, we still observe a substantial proportion of surviving CD3− CD19+ B cells (orange circle). (B) Desensitization eliminates all CD19+ CD20+ B cells from peripheral circulation (red circle, blue arrow). However, at transplantation, we observe that the CD19+ CD20+ population lose CD20 expression compared to the control group (green circle, CD19+ CD20− ). FIGURE 5 | More detailed phenotype analysis of circulating and lymph node B cells–By selection of CD3− CD19+ cells (red circle) and further analysis of phenotype, we observed a loss of naive B cells (CD27− IgD+ ) upon desensitization in the lymph node (blue circle), while CD27+ IgD− cell population still persisted (switched memory phenotype). Frontiers in Medicine | www.frontiersin.org 9 December 2021 | Volume 8 | Article 781239 Cucchiari et al. B cell Extracellular Vesicles in Kidney Transplantation i.e., alloreactive B cells resident in primary and secondary FUNDING lymphoid organs. This study has been funded by the research grant from DATA AVAILABILITY STATEMENT Instituto de Salud Carlos III (ISCIII) under the program Acción Estratégica en Salud 2016 (PI16/00115), Redes The raw data supporting the conclusions of this Temáticas de Investigación Cooperativa en Salud, REDINREN article will be made available by the authors, without (RD16/0009/0023) co-funded by ISCIII Subdirección General de undue reservation. Evaluación and Fondo Europeo de Desarrollo Regional (FEDER) Una manera de hacer Europa, and Secretaria d’Universitats i Recerca and CERCA Programme del Departament d’Economia ETHICS STATEMENT i Coneixement de la Generalitat de Catalunya (2017-SGR- The studies involving human participants were reviewed and 1331). VT received a personal grant from Fundació Catalana approved by HCB/2016/0394. The patients/participants de Trasplantament. provided their written informed consent to participate in this study. ACKNOWLEDGMENTS We thank Hernando Del Portillo PhD and Joan Segui-Barber AUTHOR CONTRIBUTIONS PhD from ISGlobal and Institut d’Investigació en Ciències de la DC was involved in study conception, laboratory experiments, Salut Germans Trias i Pujol (IGTP) for their help in the NTA data analysis, and writing of the manuscript. VT was involved analyses. We are indebted to the Cytometry and cell sorting in laboratory experiments and data analysis. JR, MR-B, facility of the Institut d’Investigacions Biomèdiques August Pi and EB-M were involved in study conception, laboratory i Sunyer (IDIBAPS) for technical help. This work has been experiment, and data analysis. ML-R and NH-G were involved developed at the Center de Recerca Biomèdica Cellex, Barcelona, in laboratory experiments. FB, JM, and AH was involved Spain. We are indebted to the Microscopy Core Facility of in study conception and data analysis. PV-A and GP were the Universitat Autónoma de Barcelona (UAB) for technical involved in data analysis and writing of the manuscript. assistance in EV electron microscopy. LP and MM were involved in data analysis. FO, JC, and FD were involved in data analysis and revision of the SUPPLEMENTARY MATERIAL final draft of the manuscript. IR was involved in study conception, data analysis, and writing of the manuscript. The Supplementary Material for this article can be found All authors contributed to the article and approved the online at: https://www.frontiersin.org/articles/10.3389/fmed. submitted version. 2021.781239/full#supplementary-material REFERENCES 7. Amrouche L, Aubert O, Suberbielle C, Rabant M, Van Huyen JD, Martinez F, et al. Long-term outcomes of kidney transplantation in patients with 1. Magee CC, Felgueiras J, Tinckam K, Malek S, Mah H, Tullius S. high levels of preformed DSA: the necker high-risk transplant program. Renal transplantation in patients with positive lymphocytotoxicity Transplantation. (2017) 101:2440–8. doi: 10.1097/TP.0000000000001650 crossmatches: one center’s experience. Transplantation. (2008) 8. Orandi BJ, Luo X, King EA, Garonzik-Wang JM, Bae S, Montgomery RA, 86:96–103. doi: 10.1097/TP.0b013e318176ae2c et al. Hospital readmissions following HLA-incompatible live donor kidney 2. Kahwaji J, Jordan SC, Najjar R, Wongsaroj P, Choi J, Peng A, et al. Six- transplantation: A multi-center study. Am J Transplant. (2018) 18:650– year outcomes in broadly HLA-sensitized living donor transplant recipients 8. doi: 10.1111/ajt.14472 desensitized with intravenous immunoglobulin and rituximab. Transpl Int. 9. Kamburova EG, Koenen HJ, Borgman KJ, ten Berge IJ, Joosten I, Hilbrands (2016) 29:1276–85. doi: 10.1111/tri.12832 LB. A single dose of rituximab does not deplete B cells in secondary lymphoid 3. Jackson AM, Kraus ES, Orandi BJ, Segev DL, Montgomery RA, Zachary organs but alters phenotype and function. Am J Transplant. (2013) 13:1503– AA, et al. closer look at rituximab induction on HLA antibody rebound 11. doi: 10.1111/ajt.12220 following HLA-incompatible kidney transplantation. Kidney Int. (2015) 10. Wen C, Seeger RC, Fabbri M, Wang L, Wayne AS, Jong AY. Biological roles 87:409–16. doi: 10.1038/ki.2014.261 and potential applications of immune cell-derived extracellular vesicles. J 4. Orandi BJ, Luo X, Massie AB, Garonzik-Wang JM, Lonze BE, Ahmed Extracell Vesicles. (2017) 6:1400370. doi: 10.1080/20013078.2017.1400370 R, et al. Survival benefit with kidney transplants from HLA-incompatible 11. McLellan AD. Exosome release by primary B cells. Crit Rev Immunol. (2009) live donors. N Engl J Med. (2016) 374:940–50. doi: 10.1056/NEJMoa15 29:203–17. doi: 10.1615/CritRevImmunol.v29.i3.20 08380 12. Bobrie A, Colombo M, Raposo G, Théry C. Exosome secretion: molecular 5. Padmanabhan A, Ratner LE, Jhang JS, Duong JK, Markowitz GS, Vasilescu mechanisms and roles in immune responses. Traffic. (2011) 12:1659– ER, et al. Comparative outcome analysis of ABO-incompatible and positive 68. doi: 10.1111/j.1600-0854.2011.01225.x crossmatch renal transplantation: a single-center experience. Transplantation. 13. Arita S, Baba E, Shibata Y, Niiro H, Shimoda S, Isobe T, et al. B cell (2009) 87:1889–96. doi: 10.1097/TP.0b013e3181a76ae1 activation regulates exosomal HLA production. Eur J Immunol. (2008) 6. De Sousa-Amorim E, Revuelta I, Blasco M, Diekmann F, Cid J, Lozano M, et 38:1423–34. doi: 10.1002/eji.200737694 al. Desensitization before living donor kidney transplantation in highly HLA- 14. Melo SA, Luecke LB, Kahlert C, Fernandez AF, Gammon ST, Kaye J, et al. sensitized patients: a single-center study. Transplant Proc. (2015) 47:2332– Glypican-1 identifies cancer exosomes and detects early pancreatic cancer. 5. doi: 10.1016/j.transproceed.2015.09.025 Nature. (2015) 523:177–82. doi: 10.1038/nature14581 Frontiers in Medicine | www.frontiersin.org 10 December 2021 | Volume 8 | Article 781239 Cucchiari et al. B cell Extracellular Vesicles in Kidney Transplantation 15. Zhang H, Huang E, Kahwaji J, Nast CC, Li P, Mirocha J, et al. Plasma exosomes transplant rejection. ACS Nano. (2017) 11:11041–6. doi: 10.1021/acsnano. from HLA-sensitized kidney transplant recipients contain mRNA transcripts 7b05083 which predict development of antibody-mediated rejection. Transplantation. 24. Feng Y, Zhong M, Zeng S, Wang L, Liu P, Xiao X, et al. Exosome- (2017) 101:2419–28. doi: 10.1097/TP.0000000000001834 derived miRNAs as predictive biomarkers for diffuse large B- 16. Tubita V, Segui-Barber J, Lozano JJ, Banon-Maneus E, Rovira J, Cucchiari cell lymphoma chemotherapy resistance. Epigenomics. (2019) D, et al. Effect of immunosuppression in miRNAs from extracellular vesicles 11:35–51. doi: 10.2217/epi-2018-0123 of colorectal cancer and their influence on the pre-metastatic niche. Sci Rep. 25. Robillard N, Wuillème S, Moreau P, Béné MC. Immunophenotype of (2019) 9:11177. doi: 10.1038/s41598-019-47581-y normal and myelomatous plasma-cell subsets. Front Immunol. (2014) 17. Haas M, Loupy A, Lefaucheur C, Roufosse C, Glotz D, Seron D, et al. The 5:137. doi: 10.3389/fimmu.2014.00137 Banff 2017 Kidney Meeting Report: Revised diagnostic criteria for chronic 26. Benameur T, Chappard D, Fioleau E, Andriantsitohaina R, Martinez active T cell-mediated rejection, antibody-mediated rejection, and prospects MC, Clere N, et al. Plasma cells release membrane microparticles for integrative endpoints for next-generation clinical trials. Am J Transplant. in a mouse model of multiple myeloma. Micron. (2013) 54–55:75– (2018) 18:293–307. doi: 10.1111/ajt.14625 81. doi: 10.1016/j.micron.2013.08.010 18. Théry C, Witwer KW, Aikawa E, Alcaraz MJ, Anderson JD, Andriantsitohaina 27. Colombo M, Giannandrea D, Lesma E, Basile A, Chiaramonte R. Extracellular R, et al. Minimal information for studies of extracellular vesicles 2018 vesicles enhance multiple myeloma metastatic dissemination. Int J Mol Sci. (MISEV2018): a position statement of the International Society for (2019) 20:3236. doi: 10.3390/ijms20133236 Extracellular Vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles. (2018) 7:1535750. doi: 10.1080/20013078.2018.1535750 Conflict of Interest: The authors declare that the research was conducted in the 19. de Menezes-Neto A, Sáez MJ, Lozano-Ramos I, Segui-Barber J, Martin- absence of any commercial or financial relationships that could be construed as a Jaular L, Ullate JM, et al. Size-exclusion chromatography as a stand-alone potential conflict of interest. methodology identifies novel markers in mass spectrometry analyses of plasma-derived vesicles from healthy individuals. J Extracell Vesicles. (2015) Publisher’s Note: All claims expressed in this article are solely those of the authors 4:27378. doi: 10.3402/jev.v4.27378 and do not necessarily represent those of their affiliated organizations, or those of 20. Böing AN, van der Pol E, Grootemaat AE, Coumans FA, Sturk the publisher, the editors and the reviewers. Any product that may be evaluated in A, Nieuwland R. Single-step isolation of extracellular vesicles this article, or claim that may be made by its manufacturer, is not guaranteed or by size-exclusion chromatography. J Extracell Vesicles. (2014) 3:23430. doi: 10.3402/jev.v3.23430 endorsed by the publisher. 21. Suárez H, Gámez-Valero A, Reyes R, López-Martín S, Rodríguez MJ, Carrascosa JL, et al. A bead-assisted flow cytometry method for the Copyright © 2021 Cucchiari, Tubita, Rovira, Ramirez-Bajo, Banon-Maneus, semi-quantitative analysis of Extracellular Vesicles. Sci Rep. (2017) Lazo-Rodriguez, Hierro-Garcia, Borràs, Ventura-Aguiar, Piñeiro, Martorell, Peri, 7:11271. doi: 10.1038/s41598-017-11249-2 Musquera, Hertig, Oppenheimer, Campistol, Diekmann and Revuelta. This is an 22. Meneghini M, Melilli E, Martorell J, Revuelta I, Rigol-Monzó E, Manonelles open-access article distributed under the terms of the Creative Commons Attribution A, et al. Combining sensitive crossmatch assays with donor/recipient human License (CC BY). The use, distribution or reproduction in other forums is permitted, leukocyte antigen eplet matching predicts living-donor kidney transplant provided the original author(s) and the copyright owner(s) are credited and that the outcome. Kidney Int Rep. (2018) 3:926–38. doi: 10.1016/j.ekir.2018.03.015 original publication in this journal is cited, in accordance with accepted academic 23. Park J, Lin HY, Assaker JP, Jeong S, Huang CH, Kurdi T, et al. practice. No use, distribution or reproduction is permitted which does not comply Integrated kidney exosome analysis for the detection of kidney with these terms. Frontiers in Medicine | www.frontiersin.org 11 December 2021 | Volume 8 | Article 781239