Graft-derived extracellular vesicles transported across subcapsular sinus macrophages elicit B cell alloimmunity after transplantation

Furong Zeng; Zhizhao Chen; Rao Chen; William J Shufesky; Mohna Bandyopadhyay; Geoffrey Camirand; Martin H Oberbarnscheidt; Mara L G Sullivan; Catherine J Baty; Mu-qing Yang; Michel Calderon; Donna Beer Stolz; Geza Erdos; Roberta Pelanda; Todd V Brennan; Sergio D Catz; Simon C Watkins; Adriana T Larregina; Adrian E Morelli

doi:10.1126/scitranslmed.abb0122

. Author manuscript; available in PMC: 2022 Mar 22.

Published in final edited form as: Sci Transl Med. 2021 Mar 17;13(585):eabb0122. doi: 10.1126/scitranslmed.abb0122

Graft-derived extracellular vesicles transported across subcapsular sinus macrophages elicit B cell alloimmunity after transplantation

Furong Zeng ^1,^2,^3,^*, Zhizhao Chen ^1,^3,^4,^*, Rao Chen ^1,^5,^*, William J Shufesky ¹, Mohna Bandyopadhyay ⁶, Geoffrey Camirand ^1,⁷, Martin H Oberbarnscheidt ^1,⁷, Mara L G Sullivan ⁸, Catherine J Baty ⁹, Mu-qing Yang ¹, Michel Calderon ⁸, Donna Beer Stolz ⁸, Geza Erdos ⁶, Roberta Pelanda ¹⁰, Todd V Brennan ¹¹, Sergio D Catz ¹², Simon C Watkins ⁸, Adriana T Larregina ^6,^7,^13,^*,^†, Adrian E Morelli ^1,^7,^*,^†,^‡

¹T.E. Starzl Transplantation Institute, Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15261, USA.

²Department of Dermatology and Rheumatology, Xiangya Hospital, Central South University, Changsha, Hunan 410008, China.

³The Third Xiangya Hospital of Central South University, Changsha, Hunan 410013, China.

⁴Hubei Key Laboratory of Medical Technology on Transplantation, Transplant Center, Institute of Hepatobiliary Diseases, Zhongnan Hospital, Wuhan University, Wuhan, Hubei 430071, China.

⁵Department of Kidney Transplantation, The Second Xiangya Hospital of Central South University, Changsha, Hunan 410011, China.

⁶Department of Dermatology, University of Pittsburgh, Pittsburgh, PA 15261, USA.

⁷Department of Immunology, University of Pittsburgh, Pittsburgh, PA 15261, USA.

⁸Department of Cell Biology, University of Pittsburgh, Pittsburgh, PA 15261, USA.

⁹Department of Medicine, Renal-Electrolyte Division, University of Pittsburgh, Pittsburgh, PA 15261, USA.

¹⁰Department of Immunology and Microbiology, University of Colorado School of Medicine, Aurora, CO 80045, USA.

¹¹Cedars-Sinai Comprehensive Transplant Center, Los Angeles, CA 90048, USA.

¹²The Scripps Research Institute, La Jolla, CA 92037, USA.

¹³McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA 15219, USA.

^‡

Corresponding author. morelli@pitt.edu

These authors contributed equally to this work.

^†

These authors contributed equally to this work as senior authors.

Author contributions: F.Z., Z.C., R.C., W.J.S., M.B., M.-q.Y., C.J.B., A.T.L., and A.E.M. performed experiments and analyzed data. F.Z., Z.C., R.C., G.C., M.H.O., C.J.B., and S.C.W. did photoconversion experiments and analysis by confocal and intravital multiphoton microscopy. M.L.G.S. and D.B.S. did the analysis by IEM. M.C. did the analysis by STED microscopy. G.E., R.P., T.V.B., and S.D.C. provided mice and technical expertise. A.E.M. and A.T.L. conceived the project, wrote the manuscript, and secured funding.

PMCID: PMC8939235 NIHMSID: NIHMS1786222 PMID: 33731430

Abstract

Despite the role of donor-specific antibodies (DSAs) in recognizing major histocompatibility complex (MHC) antigens and mediating transplant rejection, how and where recipient B cells in lymphoid tissues encounter donor MHC antigens remains unclear. Contrary to the dogma, we demonstrated here that migration of donor leukocytes out of skin or heart allografts is not necessary for B or T cell allosensitization in mice. We found that mouse skin and cardiac allografts and human skin grafts release cell-free donor MHC antigens via extracellular vesicles (EVs) that are captured by subcapsular sinus (SCS) macrophages in lymph nodes or analog macrophages in the spleen. Donor EVs were transported across the SCS macrophages, and donor MHC molecules on the EVs were recognized by alloreactive B cells. This triggered B cell activation and DSA production, which were both prevented by SCS macrophage depletion. These results reveal an unexpected role for graft-derived EVs and open venues to interfere with EV biogenesis, trafficking, or function to restrain priming or reactivation of alloreactive B cells.

INTRODUCTION

Besides the roles of innate and T cell immunity in recognition of allografts as nonself, B cells are key for transplant rejection due to their functions as antigen-presenting cells (APCs) and producers of donor-specific antibodies (DSAs) (1-3). Antibody (Ab)–mediated rejection is triggered by Abs against donor intact human leukocyte antigen (HLA) molecules and to non-HLA alloantigens and autoantigens. DSAs can be generated before transplantation in response to blood transfusions, pregnancies, or previous transplants or produced de novo, particularly in pediatric patients and medication-nonadherent adult recipients (1, 2).

Although B cell allosensitization and DSAs are major risk factors for reduced long-term graft survival (4-7), how and where alloreactive naïve B cells recognize donor HLA molecules, intact or partially degraded, is not well understood (8). In nontransplant models, it has been shown that naïve B cells detect conventional antigens alone or as immune complexes on specialized macrophages, myeloid-lineage dendritic cells (DCs), or follicular DCs (FDCs) (9-15). Nevertheless, in the case of transplantation, the graft-derived intact HLA molecules are expressed as integral glycoproteins on the surface of the donor cells, and classical studies have shown that they can also be released in a soluble format associated with lipid particles (16, 17). Because pathogenic DSAs recognize the tertiary conformation of continuous or discontinuous amino acid sequences on exposed polymorphic domains of donor HLA molecules (18), formation of immune complexes on soluble HLA molecules could hide continuous epitopes and eplets from cognate B cell receptors (BCRs).

The prevailing idea in transplantation has been that sensitization against donor intact HLA molecules is mediated by donor APCs mobilized as passenger leukocytes from the grafts to the draining secondary lymphoid tissues (dSLTs). However, it has been recently shown that after transplantation of heart, pancreatic islet, or skin allografts in mice, donor passenger leukocytes are undetectable or found at very low numbers in graft-dSLTs (19-23). The same studies demonstrated that recipient APCs cross-dressed with donor-derived extracellular vesicles (EVs) are the APCs that present the donor intact HLA molecules and its derived allopeptides to alloreactive T cells in graft-dSLTs (19-23). By contrast, the mechanism(s) by which recipient B cells detect donor intact major histocompatibility complex (MHC) molecules in graft-dSLTs, and if donor-derived EVs play a role, still remain unknown. EVs, particularly exosomes, have been shown to act as carriers for horizontal dissemination of proteins, lipids, and RNAs between cells and participate in presentation of conventional antigens (24, 25). Organ and tissue grafts release EVs, and administration of in vitro purified EVs carrying self-antigen and alloantigen elicits Ab formation (26-32).

We demonstrate here that, contrary to the prevailing dogma, B cell allosensitization and generation of de novo DSAs do not require mobilization of donor passenger leukocytes out of vascularized (heart) or nonvascularized (skin) allografts. Our in vivo findings indicate that donor MHC antigens are released cell-free from mouse and human grafts through EVs that traffic to graft-dSLTs. Ultra-structural analysis and intravital imaging revealed how the donor EVs are processed once they arrive in graft-dSLTs. First, the donor EVs are captured and concentrated by specialized macrophages lining the subcapsular sinus (SCS) in graft-draining lymph nodes (dLNs) and analog macrophages of the spleen. The donor EVs are then transported across the SCS macrophages in direction to the underlying B cell follicles, where naïve B cells recognize the donor intact MHC molecules on the EV surface. This interaction triggers calcium flux and CD86 up-regulation in alloreactive B cells, their mobilization to the B cell–T cell border, and elicitation of DSAs, which are prevented by SCS macrophage depletion. Accordingly, pharmacological inhibition of small EV biogenesis after heart transplantation in mice reduced donor EV cross-dressing of recipient APCs in graft-dSLTs, decreased the titer of DSAs in serum, and prolonged graft survival.

Despite accumulating knowledge on the biology of EVs on humoral immunity against model and microbial antigens, there is very limited information about how EVs present antigen to B cells in vivo (33-35). Our findings show a rather unexpected function for graft-derived EVs in B cell alloimmunity and open new opportunities for development of therapies to control not only Ab-mediated rejection but also T cell–mediated rejection, because alloreactive T cells require help provided by B cells primed by the same alloantigens.

RESULTS

Allosensitization in absence of mobilization of passenger leukocytes out of allografts

It is classically assumed in the field of transplantation that, in non-sensitized recipients, migration of passenger leukocytes from allografts to graft-dSLTs is key to trigger recipient sensitization against donor intact alloantigens. However, recent studies have shown in mouse models of heart, skin, or islet allografts that donor migrating leukocytes, particularly DCs, are undetectable or found at extremely low percentages in graft-dSLTs (19, 20). These studies have also shown that, in graft-dSLTs, donor intact MHC molecules are presented to directly alloreactive T cells by recipient APCs cross-dressed with donor-derived EVs (19, 20). However, the previous studies did not entirely exclude the possibility that direct allosensitization against donor intact MHC molecules was triggered by a small number of donor migratory APCs untraceable in graft-dSLTs because they are rapidly eliminated by host cytotoxic cells or because they traffic to alternative allosensitization sites (19, 20). To test the former, we transplanted CD11c–yellow fluorescent protein (YFP) C57BL/6 (B6) (H2^b) skin grafts, populated by YFP-expressing DCs, on allogeneic (H2^d) or syngeneic (CD45.1 congenic) mice. In the allogeneic combination, we confirmed that no donor YFP DCs are detectable by flow cytometry in graft-dLNs or spleen (Fig. 1A and fig. S1A) up to postoperative day (POD) 28 (19, 20). In the syngeneic (CD45.1 congenic) combination, donor YFP DCs (CD45.2) were also absent in graft-dSLTs on PODs 2 and 7 (Fig. 1B and fig. S1B). Thus, during allosensitization in the first week after surgery, the absence of donor DCs in graft-dSLTs is not due to elimination of allogeneic DCs by host cytotoxic cells, because graft-derived DCs are also undetectable in SLTs draining syngeneic grafts. Migratory DCs from syngeneic grafts became detectable in dLNs later, on PODs 14 and 28 (Fig. 1B and fig. S1B), when donor and recipient lymphatics reconnect but when skin allografts have been already rejected. If acute rejection of skin allografts is delayed by administration of the immunosuppressant FK506 (tacrolimus), then donor DCs become detectable in small numbers in graft-dLNs on PODs 14 and 28 (Fig. 1C).

Fig. 1. — (A) Flow cytometry analysis in graft-dLNs of DCs mobilized from CD11c-YFP skin allografts. (B) Detection by flow cytometry of donor DCs in graft-dLNs after transplantation of CD11-YFP skin in syngeneic (CD45.1 congenic) recipients. Right contour plots: Donor YFP DCs mobilized from the graft epidermis (CD326^Pos) and dermis (CD326^Neg CD103^Pos). (C) Quantification by flow cytometry of donor DCs in graft-dLNs after transplantation of CD11c-YFP skin in syngeneic (syn) or allogenic (allo) recipients treated or not with FK506 (FK). ND, not detected. Numbers above bars indicate numbers of donor DCs. (D and E) Kaplan-Meier analysis of survival of CCR7^KO and wt B6 skin (D) or heart (E) allografts in wt BALB/c mice. Numbers of recipients are in parentheses. (F and G) ELISpot analysis on POD 7 of the T cell response against donor intact MHC molecules (direct pathway) in graft-dLNs of BALB/c mice transplanted with wt or CCR7^KO skin allografts (F) or in spleens of BALB/c mice grafted with wt or CCR7^KO cardiac allografts (G). IFN-γ, interferon-γ. (H and I) Titer of DSAs analyzed by flow cytometry in serum of BALB/c mice before surgery and on successive PODs after transplantation of wt or CCR7^KO skin (H) or heart (I) allografts. In (A) and (B), numbers in dot or contour plots are cell percentages. (A to C) Six mice per group. In (F) to (I), each dot represents a recipient. Results in (F) to (I) were analyzed by one-way ANOVA, followed by Tukey-Kramer multiple comparison test. Error bars denote means ± SD. NS, not significant. **P < 0.01 and ***P < 0.001.

Next, we tested whether donor APCs trigger allosensitization by trafficking to other allosensitization sites than those analyzed in previous studies (19, 20). We transplanted CCR7^KO allografts in which donor leukocytes are unable to leave the grafts (36). First, we validated in our models that CCR7^KO skin and heart allografts have similar content of passenger leukocytes as their wild-type (wt) counterparts (fig. S2, A to E), that CCR7 deficiency severely impairs migration of allogeneic DCs to dLNs (P = 0.0008; fig. S2F), and that donor DCs are unable to migrate to the dermal lymphatics in CCR7^KO skin allografts (fig. S3A). We also confirmed that donor passenger leukocytes are undetectable by polymerase chain reaction (PCR) in LNs draining either wt or CCR7^KO skin allografts (fig. S3B) and that donor DCs are unable to mobilize to lymphatic vessels in CCR7^KO cardiac allografts (fig. S3C). CCR7^KO and wt skin and heart allografts were rejected at similar tempo (Fig. 1, D and E) and elicited comparable allosensitization of directly alloreactive T cells [assessed by enzyme-linked immune absorbent spot (ELISpot)] and B cells (measured by titration of DSAs in serum) in graft-dSLTs (Fig. 1, F to I). Thus, in the transplantation models tested, migration of donor leukocytes from nonvascularized or vascularized allografts to SLTs was not necessary for allosensitization of B cells and directly alloreactive T cells.

The assumption that migration of donor DCs to graft-dSLTs is key for allosensitization is based on previous findings in mice that the density of epidermal Langerhans’ cells in skin allografts decreases during the first three PODs, which we confirmed (P < 0.0001; Fig. 2A) (37). Because we did not detect donor DCs in graft-dLNs (Fig. 1A), we wondered whether the donor DCs are retained within the skin allografts. During the first three PODs, donor DCs accumulated in the graft dermis next to or within lymphatic vessels (P = 0.0023; Fig. 2, B and C).

Fig. 2. — (A) Planimetric view of donor Langerhans cells on epidermis of skin allografts and quantification on successive PODs. Images representative of six grafts per variable. Magnification, ×100. (B) Horizontal view of donor DCs (red) in dermal laminas of skin allografts. On POD 1, donor DCs accumulated inside lymphatics (arrows, inset). Images are representative of four grafts per group. Quantification by flow cytometry of donor DCs in dermal cell suspensions of skin allografts. Three to six grafts per group. Magnification, ×200 and ×400. (C) Vertical section of a skin allograft (POD 3) showing a dermal lymphatic vessel (blue) clogged with donor cells (green), with dendritic morphology, some coexpressing CD326 (inset). Dotted line indicates epidermal-dermal junction. Image representative of four allografts. X200, X400. EpCAM, Epithelial Cell Adhesion Molecule. (D) ImageStream of recipient DCs and B cells from LNs draining skin allografts on POD 2 cross-dressed with donor H2K^b and H2D^b. Magnification, ×60; 20,000 cells. (E) Quantification by ImageStream in graft-dLNs of recipient DCs and B cells cross-dressed with donor H2K^b and H2D^b after transplantation of allogeneic or syngeneic skin set as background (BKGD) staining. Each dot represents a recipient. (F) IEM images of recipient DCs and B cells, FACS-sorted from graft-dLNs on POD 2, cross-dressed with EVs carrying donor-derived H2K^b/IA^b and CD63 (inset). N, nucleus. Magnification, ×2,500 to 10,000. Representative images of 60 to 80 immunogold-labeled cells. In (A), (B), and (F), results were analyzed by one-way ANOVA, followed by Tukey-Kramer multiple comparison test. Error bars represent means ± SD. **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Allografts transfer donor alloantigen to graft-dSLTs via EVs

It has been previously shown that after transplantation of skin or heart allografts, recipient APCs in graft-dSLTs become cross-dressed with donor-derived EVs (19, 20), a phenomenon that we confirmed here in the skin transplant model by analysis with ImageStream (Fig. 2, D and E) and immunoelectron microscopy (IEM) (Fig. 2F). These recipient APCs cross-dressed with donor EVs in graft-dSLTs could result from acquisition by recipient APCs of donor-derived EVs transported by graft-infiltrating leukocytes (GILs) that migrated from the grafts to the dSLTs, a subset of GILs cross-dressed within the graft with donor-derived EVs that then homed in graft-dSLTs, or recipient APCs resident in dSLTs that acquire in situ donor EVs released directly by the allografts. To determine the contribution of GILs on donor MHC cross-dressing of recipient leukocytes in graft-dSLTs, we transplanted BALB/c skin allografts in mice expressing ubiquitously the Kikume Green-Red (KikGR) fluorescent photoconvertible protein and exposed them daily on the grafts to violet light to shift the fluorescence from green to red in GILs (Fig. 3, A and B) (38). In this model, those GILs that migrate from the skin grafts to the dLNs are detectable by flow cytometry as photoconverted cells (Fig. 3C). Previous studies have shown that after transplantation of skin allografts in mice, donor-MHC cross-dressing in graft-dLNs reaches its peak between 3 and 7 days after surgery (19, 20). On PODs 3 and 7, donor MHC was detectable by flow cytometry analysis on LN-resident leukocytes but not on photoconverted leukocytes mobilized from the allografts to dLNs (P < 0.0001; Fig. 3, D and E). These findings indicate that recipient APCs in graft-dSLTs do not acquire donor intact MHC molecules from donor MHC cross-dressed GILs that left the graft and circulated through graft-dSLTs and that they are not a subset of donor MHC cross-dressed GILs that abandoned the graft and homed in graft-dSLTs.

Fig. 3. — (A) CMV-Cre X ROSA26-loxP-stop-loxP-KikGR F1 mice expressing KikGR-green in ≥99% of PBMCs, measured by flow cytometry, were used as recipients. (B) Flow cytometry analysis (POD 3) of photoconversion of KikGR-green into KikGR-red in GILs within BALB/c skin grafts transplanted in B6 KikGR mice. Grafts were left untreated or exposed to violet light on PODs 1 and 2. One representative of six grafts per variable. (C) Quantification by flow cytometry of photoconverted leukocytes in LNs draining skin allografts left untreated or exposed to violet light on PODs 1 and 2 for POD 3 analysis, or PODs 1 to 6 for POD 7 analysis. Each dot corresponds to graft-dLNs from individual recipients. (D) Flow cytometry analysis on PODs 3 and 7 of donor MHC cross-dressing on photoconverted GILs and nonphotoconverted leukocytes in graft-dLNs. Donor MHC cross-dressing was detected on nonphotoconverted leukocytes (KikGR-red^Neg cells within the red gates). Dot plots correspond to one set of graft-dLNs representative of six for POD 3 and seven for POD 7. (E) Quantification by flow cytometry on PODs 3 and 7 of percentages of donor MHC cross-dressing on photoconverted and nonphotoconverted leukocytes in LNs draining skin allografts exposed to violet light on PODs 1 and 2 for POD 3 analysis or PODs 1 to 6 for POD 7 analysis. Each dot corresponds to graft-dLNs from different recipients. In (C) and (E), comparisons were performed using two-tailed Student’s t test. Error bars denote means ± SD. *P < 0.05, ***P < 0.001, and ****P < 0.0001.

We next analyzed whether allografts deliver cell-free donor antigen to dSLTs. One day after transplantation of skin allografts (H2^b), we detected donor H2 (IA^b) content within CD169 macrophages lining the SCS and transverse sinus of dLNs (Fig. 4A and fig. S4A). Donor IA^b was also detected associated with B cells and FDCs in the underlying B cell follicles (fig. S4A). As controls, IA^b was negative in non-dLNs of skin allograft recipients, and in LNs draining syngeneic grafts (fig. S4A). Traffic of cell-free donor IA^b was confirmed in LNs draining MHC class II (IA^b) fused to the enhanced green fluorescence protein (EGFP) or CCR7^KO skin allografts (fig. S4, B and C).

Fig. 4. — (A) Detection of donor IA^b in SCS macrophages (inset) in graft-dLN after transplantation of skin allografts. Images representative of six recipients. Magnification, ×200. (B) IEM image of SCS of graft-dLN containing EVs carrying donor H2K^b/IA^b and CD63 (red arrows, inset). White arrow indicates CD169 expression by SCS macrophages. Diagram shows image interpretation. (C) SCS macrophages (CD169, white arrows) containing EVs bearing donor H2K^b/IA^b and CD63 (red arrows, inset) within a system of interconnected vesicles or channels (asterisks). Magnification, x20,000 and x80,000. Size of donor EVs internalized by SCS macrophages. (D) Sequence of allogeneic EVs captured by SCS macrophages in a pLN. Numbers indicate minutes after injection. (E) Two-photon microscopy of allogeneic EVs captured by SCS and medullary macrophages in a pLN. Dotted line indicates cortex-medulla junction. (F) Uptake of footpad-injected EVs by leukocytes in draining pLNs as measured by flow cytometry. Horizontal dotted lines indicate ex vivo uptake of exogenous EVs by bystander CD45.1 leukocytes. (G) STED microscopy of allogeneic EVs crossing a system of interconnected vesicles or tunnels filled with lymph tracer (blue) within pLN SCS macrophages. Line indicates LN capsule. (H) Sequence by STED microscopy in a pLN of transport across an SCS macrophage of allogeneic CM-Dil-EVs (inset). Arrows indicate directional passage of CM-Dil^Pos material. One representative of two pLNs. (A to E) Images representative of graft-dLNs from three to four recipients. In (D) to (H), an equivalent of 55 × 10⁸ allogeneic EVs (87 ± 49 nm in size) in 30 μl of PBS was injected per footpad. In (F), results were analyzed by one-way ANOVA, followed by Tukey-Kramer multiple comparison test. Error bars denote means ± SD. **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Next, we determined the format by which the cell-free donor antigen is delivered to dSLTs. IEM analysis revealed that passage of donor antigen from skin allografts was mediated via EVs of about 82.3 nm carrying donor H2K^b and IA^b and occasionally the EV-associated marker CD63 (Fig. 4, B and C). They were detected as individual EVs in the SCS lumen or inside SCS CD169 macrophages that concentrated the EVs into clusters within a system of interconnected vesicles or tunnel-like structures that run across the SCS macrophages (Fig. 4C and fig. S5, A and B). To confirm these findings, we transplanted skin allografts encoding ubiquitously expressed Tag Red Fluorescent Protein (TagRFP) linked to the EV-associated marker CD63 (fig. S5, C and D). On POD 3, RFP was detected inside SCS CD169 macrophages of graft-dLNs (movie S1). Shuttling of donor MHC molecules to dSLTs via EVs also occurs after transplantation of heart allografts, where donor IA^b was detected in splenic marginal zone CD169 macrophages, DCs, and FDCs (fig. S6, A to C). In vitro analysis by flow cytometry of small EVs released by bone marrow (BM)–derived DCs from B6 mice revealed that intact MHC molecules (H2K^b and IA^b) are not only transported exclusively by CD63^Pos EVs but also transported by CD63^Neg EVs expressing the tetraspanins CD9 or CD81 (fig. S7).

Donor EVs are rapidly captured by SCS macrophages and other cell subsets in dLNs

To visualize how allogeneic EVs disseminate in dSLTs, we injected CellTracker CM-DiI (CM-DiI)–labeled allogeneic EVs purified from BM-derived DC culture supernatants into the footpads of mice and monitored their arrival in popliteal LNs (pLNs) by intravital two-photon microscopy. The amount of EVs administered (~55 × 10⁸ EVs) was equivalent to that released by one skin (ear) allograft during 2-day culture (fig. S8, A and B). Within 30 min, SCS CD169 macrophages captured the incoming EVs and concentrated them in punctate areas within the cell (Fig. 4, D and E, and movie S2, parts I and II). The lymph-borne EVs were also trapped by medullary macrophages and DCs (Fig. 2E and movie S2, parts II and III). Flow cytometry analysis of draining pLNs confirmed that the lymph-borne allogeneic EVs were trapped mainly by SCS and medullary macrophages and, to a lesser extent, by DCs and B cells (Fig. 4F and fig. S8C). Addition of bystander CD45.1 congenic LN cells at the time of LN cell isolation for flow cytometry analysis confirmed that the subcutaneously injected EVs were captured in vivo by the host’s CD45.2 leukocytes (Fig. 4F and fig. S8C).

Donor EVs traffic across SCS macrophages in dLNs

Because graft-derived EVs carried donor MHC molecules on the surface, we investigated whether donor EVs participate in allosensitization of donor-reactive B cells. Previous studies tracking the fate of lymph-borne immune complexes, opsonized antigen, and pathogens arriving in LNs revealed that relatively small amounts of the antigenic particles are captured by SCS macrophages and displayed for initial cognate recognition by follicular B cells (9-11, 39). In the skin transplant model used here, we showed that the donor alloantigen is carried by lymph-borne EVs that arrive in the SCS of graft-dLNs where the donor EVs enter the apical zone of SCS macrophages (Fig. 4, B to D). Next, we analyzed how the donor EVs traffic across the SCS floor to be displayed and recognized by the underlying follicular B cells. To do that, we analyzed explanted pLNs by super-resolution stimulated emission depletion (STED) microscopy 2.5 hours after footpad injection of CM-DiI–labeled allogeneic EVs and a lymphatic fluid tracer, in mice injected via footpad with Alexa Fluor 488 (AF488)–CD169 Ab to label the SCS macrophages. Three-dimensional reconstruction revealed that the EVs are channeled through a tunnel-like system of interconnected vesicles that was filled by the lymphatic fluid tracer and traversed the SCS macrophages (Fig. 4G and movie S3, part I). Time-lapse analysis by STED microscopy indicated that the donor EVs are transported longitudinally and unidirectionally across this system (Fig. 4H and movie S3, part II).

Alloreactive B cells recognize donor EVs displayed by SCS macrophages in dLNs

Little is known regarding how B cells detect donor antigen after transplantation. Our findings indicate that shedding of donor antigen independently of donor passenger cell migration is sufficient for eliciting de novo DSAs (Fig. 1, H and I) and that donor EVs are captured by specialized macrophages in dSLTs (Fig. 4, D to H). In LNs, antigen phagocytosed by medullary macrophages is rapidly degraded, whereas immune complexes, particulate antigen, or viruses trapped by SCS macrophages are presented to B cells (9-11, 40). Therefore, we investigated by intravital two-photon microscopy if B cells recognize allogeneic EVs displayed by SCS macrophages. BALB/c mice injected intravenously with CellTracker Blue CMAC (CMAC)-labeled 3-83 immunoglobulin (Ig) B cells, expressing a BCR that binds H2K^k (41), were footpad-injected with CM-DiI-C3H (H2K^k) EVs and AF488-CD169 Ab, the latter to label in vivo the SCS macrophages. Two hours after EV injection, 3-83 Ig B cells were detected probing SCS macrophages in pLNs and remained confined for longer times next to those SCS macrophages loaded with allogeneic EVs, compared to 3-83 Ig B cells that did not interact with SCS macrophages or probed SCS macrophages without allogenic EVs (Fig. 5, A to C, and movie S4). To ascertain whether the 3-83 Ig B cell arrest was allospecific, we conducted similar experiments in BALB/c mice injected intravenously with CMAC-labeled 3-83 Ig B cells and control carboxyfluorescein diacetate succinimidyl ester (CFSE)–labeled wt BALB/c B cells. Two hours after CM-DiI-C3H EV injection, 3-83 Ig B cells accumulated below the SCS in higher numbers (P = 0.0035), exhibited lower mean velocity (P = 0.0007) with higher arrest coefficient (P < 0.0001), and made longer contacts with C3H EV–laden SCS macrophages (P < 0.0001), than control B cells (Fig. 5, D and E, and movie S5, parts I and II). No significant differences in those parameters were detected between 3-83 Ig B cells and control B cells after injection of syngeneic EVs (mean velocity, P = 0.3864; arrest coefficient, P = 0.2877; distance to SCS floor, P = 0.0561) (fig. S9A and movie S5, parts III and IV).

Fig. 5. — (A) Image of a pLN showing tracks of alloreactive B cells contacting SCS macrophages with allogeneic EVs. (B) Instant velocities of individual B cell tracks from (A). (C) Mean velocities and arrest coefficients of alloreactive B cells contacting SCS macrophages with allogeneic EVs. (D) Cell tracks of B cells in proximity to SCS macrophages with allogeneic EVs. (E) Parameter comparison by two-photon microscopy between alloreactive and control B cells in pLNs, after footpad injection of allogeneic EVs. (F) Sequence of calcium flux in an alloreactive B cells contacting SCS macrophages with allogeneic EVs in pLNs. (G) Mobilization of alloreactive B cells to the B cell–T cell border of pLNs, after footpad injection of allogeneic EVs. X200. (H) Sequence of passage of allogeneic EVs to alloreactive B cells in pLNs (arrow, inset). (I and J) Flow cytometry analysis (I) and quantification (J) in pLNs of binding of CM-DiI-EVs to intravenously (i.v.) injected alloreactive (CMAC) or control (CFSE) B cells or endogenous B cells. (K) Flow cytometry analysis in pLNs of CD86 by intravenously injected alloreactive B cells after footpad injection of allogeneic or syngeneic CM-DiI-EVs. In (A) to (K), an equivalent of 55 × 10⁸ allogeneic EVs (85 ± 39 nm in size) or syngeneic EVs (109 ± 49 nm in size) in 30 μl of PBS was injected per footpad. (A to K) Results are representative of three to nine pLNs per condition. In (J) and (K), each dot represents a pLN. Results analyzed by two-tailed Student’s t test and one-way ANOVA, followed by Tukey-Kramer multiple comparison test. Error bars denote means ± SD. **P < 0.01, ***P < 0.001, and ****P < 0.0001.

We next investigated whether the donor-reactive B cell arrest observed in pLNs during B cell contact with SCS macrophages loaded with footpad-injected allogeneic EVs also occurs with SCS macrophages of LNs draining allografts. To do so, we grafted BALB/c mice with C3H skin on the upper back and intravenously injected them with CellTrace Violet-3-83 Ig B cells and control CellTrace Far Red–labeled wt BALB/c B cells the following day. AF488-CD169 Ab was also administered subcutaneously under the graft to label SCS macrophages located in the lymphoid lobules that specifically drain the allograft in the brachial (graft-draining) LN. The next day, analysis by two-photon microscopy of the graft-dLNs revealed that the donor-reactive B cells are retained for significantly longer times in proximity to the AF488-CD169 Ab-labeled SCS macrophages, as compared to control B cells (mean velocity, P = 0.0006; arrest coefficient, P = 0.0089; fig. S9B and movie S6, parts I and II). As controls, 3-83 Ig B cells and wt BALB/c B cells exhibited similar mean velocities and arrest coefficients after interaction with SCS macrophages in LNs draining syngeneic grafts imaged on POD 2 (mean velocity, P = 0.2620; arrest coefficient, P = 0.5792; fig. S9C and movie S6, parts III and IV).

To further analyze whether donor-reactive B cells established stable contacts with SCS macrophages loaded with graft-derived EVs, we conducted similar experiments in BALB/c mice transplanted with B6 skin expressing H2K^b, an MHC class I allele that is recognized by 3-83 Ig B cells (42), and encoding TagRFP-CD63 under control of CD11c promoter, the latter to direct RFP expression specifically to the EVs released by the donor DCs retained in the skin allografts. Two days after surgery, RFP content was detected in SCS macrophages of graft-dLNs after transplantation of skin allografts from CD11c-Cre/LoxP- RFP-CD63 B6 mice and was absent in SCS macrophages draining skin allografts from CD11c-Cre^Neg littermate controls (movie S7, part I). On POD 2, 3-83 Ig B cells were detected in close contact and for prolonged time with SCS macrophages loaded with RFP^Pos content in the graft-dLNs (movie S7, part II). As specificity control of the graft-derived EV labeling for the latter experiment, similar results were obtained when RFP signal was undetectable in SCS macrophages of LNs draining skin grafts obtained from CD11c-Cre^Neg littermate controls (movie S7, part III).

Presentation of graft-derived EVs by SCS macrophages initiates donor-reactive B cell activation

Next, we asked whether recognition by B cells of allogeneic MHC on EVs presented by SCS macrophages triggers B cell activation. BALB/c mice intravenously receiving CMAC-labeled 3-83 Ig B cells loaded with the calcium probe Fluo-4 acetoxymethyl (AM) ester were injected via footpad with CM-DiI-C3H EVs or control syngeneic EVs and the pLNs examined by intravital two-photon microscopy. Two hours after EV injection, 3-83 Ig B cells emitted flashes upon contact with C3H EV–laden SCS macrophages, a phenomenon undetected in controls injected with syngeneic EVs (Fig. 5F and movie S8). 3-83 Ig B cells mobilized to the B cell–T cell border in pLNs 24 hours after injection (footpad) of C3H EVs, but not after administration of syngeneic EVs, which is indicative of allospecific B cell activation (Fig. 5G).

Next, we analyzed whether the allogeneic EVs concentrated and mobilized across SCS macrophages are also transported by B cells throughout the B cell follicle. After injection (footpad) of CM-DiI-C3H EVs, we detected in pLNs, passage of CM-DiI content from SCS macrophages to CMAC-labeled 3-83 Ig B cells (Fig. 5H and movie S9, part I). 3-83 Ig cells carrying CM-DiI⁺ material were also found within the B cell follicle (12 of 434 cells; movie S9, part II), and a phenomenon not detected in control CFSE-labeled wt B cells coinjected intravenously (0 of 804 cells). Flow cytometry analysis revealed that, in vivo, more 3-83 Ig B cells bound allogeneic (C3H) EVs than syngeneic EVs (Fig. 5, I and J). It also demonstrated that 3-83 Ig B cells recognize allogeneic EVs via the BCR, because injection of allogenic EVs but not syngeneic EVs led to CD86 up-regulation (Fig. 5K). Binding was dependent on the physical properties of the vesicles because it diminished after administration of disrupted EVs (Fig. 5K). The findings that small percentages of control B cells (endogenous or injected intravenously) capture both allogeneic and syngeneic EVs to similar extent and that a small percentage of 3-83 Ig B cells take up syngeneic EVs (Fig. 5, I and J) indicate that B cells also bind allogeneic EVs via noncognate mechanisms that are less efficient than cognate recognition.

To test the relevance of SCS macrophages in B cell allosensitization in response to donor EVs, we injected C3H EVs into footpads of BALB/c mice depleted of SCS macrophages by prior footpad injection of low-dose clodronate liposomes (fig. S10). In pLNs depleted of SCS macrophages, intravenously injected 3-83 Ig B cells exhibited significantly less uptake of allogeneic EVs (P = 0.0019), reduced up-regulation of CD86 (P = 0.0012; Fig. 6, A to D), and did not mobilize to the B cell–T cell border (Fig. 6, E and F). Accordingly, depletion of SCS macrophages decreased serum concentrations of DSAs elicited by footpad-injected allogeneic EVs (Fig. 6G).

Fig. 6. — (A and B) Representative flow cytometry analysis (A) and quantification (B) in pLNs of binding of syngeneic and allogeneic EVs (CM-DiI–labeled, footpad-injected) by CMAC-labeled, intravenously injected alloreactive B cells in presence or absence of SCS macrophages. SCS macrophages were depleted by administration of low-dose clodronate liposomes via footpad injection. (C and D) Representative flow cytometry analysis (C) and quantification (D) in pLNs of CD86 expression by CMAC-labeled, intravenously injected alloreactive B cells in response to syngeneic or allogeneic EVs (CM-DiI–labeled, footpad-injected), in presence or absence of SCS macrophages. (E and F) Mobilization to the B cell–T cell border in pLNs of CFSE-labeled, intravenously injected alloreactive B cells after footpad injection of syngeneic or allogeneic EVs in presence of SCS macrophages or after depletion. B cell migration was assessed 20 hours after EV injection. Images representative of eight LNs per condition. (G) Detection by flow cytometry of DSAs in serum of mice before and on consecutive days after footpad injection of syngeneic or allogeneic EVs. Mice were left untreated or injected once with control or low-dose clodronate liposomes (footpad and intravenous) 5 days before EV injection. Dots represent individual mice. In (B), (D), and (F), each dot represents pLNs pooled from one mouse. In (A) to (G), an equivalent of 55 × 10⁸ EVs (85 ± 39 nm in size) or syngeneic EVs (109 ± 49 nm in size) in 30 μl of PBS was injected per footpad. Results were analyzed by one-way ANOVA, followed by Tukey-Kramer multiple comparison test. Error bars denote means ± SD. **P < 0.01 and ***P < 0.001.

We showed that long after mouse skin and heart allografts are fully rejected (between PODs 8 and 13; Fig. 1, D and E), the titer of DSAs in serum continued increasing (Fig. 1, H and I). Thus, we investigated whether donor intact MHC molecules are retained by the recipient SLTs for potential B cell allorecognition after the allografts are fully rejected. Forty-five days after transplantation of B6 skin or heart allografts, donor intact MHC molecules were detected in small foci on recipient FDCs in B cell areas of the respective graft-dSLTs (fig. S11, A and B). Analysis by super-resolution STED microscopy suggested that the donor MHC molecules were delivered to FDCs through small EVs (fig. S11A). We confirmed that 45 days after transplantation of skin or heart allografts in mice, residual donor passenger leukocytes are an unlikely source of donor intact MHC molecules for alloreactive B cells, because donor cells were undetectable by PCR in the graft-draining SLTs on POD 45 (fig. S11C).

Inhibition of EV biogenesis diminishes generation of de novo DSAs

Because our findings may have application to control B cell and T cell allosensitization after transplantation, we tested whether pharmacological inhibition of biogenesis of small EVs, including exosomes, with the neutral sphyngomyelinase inhibitor GW4869 had an effect on the titer of DSAs in serum. Treatment of BALB/c mice with GW4869 for up to 1 month did not have detectable harmful effects on the animals’ well-being or on the architecture and leukocyte composition of SLTs in general (fig. S12, A to E). Exposure to GW4869 did not affect the ability of T cells or B cells to recognize alloantigens, because GW4869-treated and vehicle dimethyl sulfoxide (DMSO)–treated BALB/c mice developed similar T cell–mediated allogeneic delayed-type hypersensitivity responses in the skin and titer of DSA in serum (fig. S12, F and G) in response to allogeneic splenocytes injected intradermally in the ear skin or intraperitoneally, respectively. In serum, GW4869 administration reduced the number of circulating EVs (mean size range, 80.2 to 134.0 nm between samples) (fig. S12H).

Because generation and release of EVs occur quickly, we pretreated donor mice with GW4869 on PODs −5, −3, and −1 to minimize production or secretion of donor EVs once the allografts were in place. Recipients were treated each other day beginning on POD 1. GW4869 administration prolonged cardiac allograft survival in mice (P = 0.0004; Fig. 7A). Nevertheless, because GW4869 may exert off-target effects, we assessed its effect on donor MHC cross-dressing of recipient APCs and direct alloreactivity. GW4869 reduced the percentages of recipient APCs cross-dressed with donor H2K^b and H2D^b antigen in graft-dSLTs (recipient DCs, P = 0.0353; recipient B cells, P = 0.0102; Fig. 7, B and C) and their capacity to present donor intact IA^d acquired in vivo to directly alloreactive T cell receptor transgenic 4C CD4 T cells (Fig. 7D). It also decreased the titer of de novo DSAs in recipient serum (Fig. 7E). Rab27a has been shown to participate in the release of small EVs (43). However, allografts deficient in Rab27a did not exhibit prolonged survival (P = 0.1400) or augment the beneficial effects of GW4869 (P = 0.7999; fig. S13A). In our hands, GW4869 treatment was more efficient than Rab27a deficiency at decreasing the yield of small EVs released by BM DCs (81.8 ± 7.8% versus 37.8 ± 15.8% reduction, P = 0.0047; fig. S13B). This suggests that grafts deficient in Rab27a still release enough EVs or passenger leukocytes to trigger allosensitization or likely that EVs other than donor-derived exosomes also participate in allosensitization in our transplant model.

Fig. 7. — (A) Kaplan-Meier analysis of survival of B6 cardiac allografts in mice treated short or long term via intraperitoneal (i.p.) injection of GW4869 or vehicle (DMSO). Number of recipients and graft survival days are in parenthesis. (B) Representative analysis by ImageStream of recipient splenic APCs cross-dressed with donor H2K^b and H2D^b, 2 days after transplantation of B6 CD45.1 cardiac allografts. CD63 colocalized in areas containing H2K^b H2D^b. Magnification, X60, 40,000 cells. (C) Assessment by ImageStream of recipient total leukocytes and APCs cross-dressed with donor H2K^b and H2D^b in splenocytes of BALB/c mice grafted with B6 CD45.1 hearts and treated with GW4869 or vehicle. Splenocytes of naïve BALB/c mice were included to set background (BKGD) staining (dotted lines). Each dot represents a mouse or graft recipient. No Tx, no transplant. (D) Flow cytometry analysis of division (CFSE dilution) and activation (CD44^High) of CFSE-labeled T cell receptor transgenic 4C CD4 T cells, specific for IA^d, in response to ex vivo stimulation with recipient (YFP^Pos) DCs FACS-sorted from spleens of CD11c-YFP B6 mice untreated (negative control) or grafted 3 days prior with BALB/c (H2^d) hearts and treated with GW4869 or DMSO. Plots are representative of four transplants per group. (E) Effect of administration GW4869 or DMSO on DSA titers in serum of BALB/c mice grafted with B6 hearts. Each dot represents a mouse. Results in (C) and (E) were analyzed by one-way ANOVA, followed by Tukey-Kramer multiple comparison test. Error bars denote means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Donor EVs released by human grafts accumulate in human SCS macrophages in graft-dLNs

To evaluate whether our results are relevant to human transplants, we investigated in humanized mice (huMice) if human skin grafts shed cell-free donor antigen via EVs and if graft-derived EVs are captured in vivo by human SCS macrophages in graft-dSLTs. To address that, human HLA-A2 skin grafts were transplanted on HLA-A2^Neg huMice, a model in which graft-dLNs are readily detectable on POD 3 (fig. S14). The huMice used as graft recipients had ≥75% of human chimeric leukocytes (Fig. 8A). The floor of the SCS of these graft-dLNs was covered by human SCS macrophages intermingled with mouse lymphatic endothelium [mouse lymphatic vessel endothelial receptor-1 (LYVE-1)^Pos] (fig. S14) and remaining mouse SCS macrophages (fig. S15A). On POD 3, donor-derived (HLA-A2^Pos) passenger leukocytes were undetectable by immunofluorescence microscopy in graft-dLNs of HLA-A2^Neg huMice. However, similar to our findings in the mouse transplant models, donor-derived (HLA-A2^Pos) cell-free content was found within the human SCS macrophages of graft-dLNs (Fig. 8B and fig. S15B), the latter detected with an anti-human CD169 Ab that do not cross-react with the mouse CD169 analog (fig. S15C). By IEM analysis, donor-derived HLA-A2^Pos EVs were detected free in the SCS lumen or attached to or internalized by recipient human SCS CD169 macrophages (Fig. 8C and fig. S16A). Some of the HLA-A2^Pos EVs coexpressed low amounts of human CD63 (Fig. 8C and fig. S16A) labeled with an anti-human CD63 rabbit monoclonal Ab that does not recognize mouse CD63 (fig. S16B). ImageStream analysis of LNs draining human HLA-A2^Pos skin grafts revealed the presence of donor HLA-A2 antigen on punctate areas on human DCs and B cells of recipient origin (Fig. 8D). The cell surface areas cross-dressed with donor HLA-A2 antigen occasionally bore human CD63 (Fig. 8D) detected by an anti-human CD63 mouse monoclonal Ab that does not cross-react with mouse CD63 (fig. S16B). HLA-A2 staining was negative in control graft-dLNs of huMice transplanted with HLA-A2^Neg human skin and in non–(graft)-draining (mesenteric) LNs of huMice transplanted with HLA-A2^Pos skin (Fig. 8E). Thus, human grafts also release in vivo EVs that carry intact alloantigen to graft-dLNs.

Fig. 8. — (A) Dot plots indicate percentages of human leukocyte chimerism in huMice (spleen) analyzed by flow cytometry. (B) Detection by microscopy of donor HLA-A2 in human SCS CD169 macrophages (insets) in LNs of HLA-A2^Neg huMice draining human HLA-A2^Pos skin grafts on POD 3. X200, X400. Images representative of six recipients. (C) IEM images of ultrathin cryosections of LNs of HLA-A2^Neg huMice, draining human HLA-A2 skin grafts isolated on POD 3 depicting donor (HLA-A2^Pos) EVs (red arrows, insets) in the SCS, next to, or internalized by SCS macrophages of human origin (white arrows indicate human CD169 expression). Images representative of four graft-dLNs. h, human. Magnification, X80,000, X200,000. (D) ImageStream of recipient human APCs cross-dressed with donor HLA-A2 from LNs of HLA-A2^Neg huMice draining HLA-A2 human skin grafts isolated on POD 3. X60, 40,000 cells. (E) ImageStream quantification of human APCs cross-dressed with donor HLA-A2 in HLA-A2^Neg huMice transplanted with HLA-A2^Pos or HLA-A2^Neg (control) human skin. Irrel, irrelevant. Each dot represents a LN from a different huMice. Results in (A) and (E) were analyzed by two-tailed Student’s t test and one-way ANOVA, followed by Tukey-Kramer multiple comparison test, respectively. Error bars denote means ± SD. **P < 0.01.

DISCUSSION

The present study gives insights into the mechanism of B cell allosensitization and de novo generation of DSAs. We provide ultra-structural, in vivo, and functional evidence on how donor-reactive naïve B cells in SLTs detect donor intact MHC molecules expressed on the surface of nonmigratory and migratory cells of allografts. Here, we demonstrate in mice that migration of passenger leukocytes out of nonvascularized skin or vascularized cardiac grafts is not required for allosensitization of B cells or directly alloreactive T cells or elicitation of acute rejection. Moreover, in the skin transplant model, passenger leukocytes were undetectable by flow cytometry in graft-dLNs during the first week after surgery when allosensitization occurs, which agrees with previous observations (19, 20, 44, 45). Our results indicate that this is not due to rapid elimination of nonself-migratory passenger cells by recipient cytotoxic leukocytes (44, 46-48). It is nevertheless coincidental with the transient lack of continuity during the first week after surgery between the severed ends of the donor and recipient lymphatic vessels, the main route of mobilization of passenger leukocytes from nonvascularized skin allografts to dLNs. It has been previously shown that early after skin transplantation, donor Langerhans cells leave the graft epidermis, which gives the appearance that they migrate out of the allografts to the dSLTs (37). By contrast, we demonstrate that the donor DCs remain trapped near or inside the severed lymphatic vessels of the graft dermis. Our results also reveal that in the cardiac allograft model, in which passenger leukocytes migrate in low numbers to the spleen and mediastinal LNs (19, 49, 50), mobilization of passenger leukocytes out of the grafts is not required for B cell or T cell allosensitization or elicitation of acute rejection of the transplant.

If migration of passenger leukocytes is not essential for transport of donor MHC molecules to the dSLTs and sensitization against donor MHC, then another possibility is that recipient GILs cross-dressed with donor MHC antigen in the grafts and then migrate to the dSLTs (51, 52). GILs have been shown to traffic from retransplanted skin allografts to dLNs and present donor peptides via cross-priming to CD8 T cells (45). Thus, we speculated that GILs mobilized to dLNs could also present donor MHC molecules acquired by cross-dressing to naïve B cells. Alternatively, migrating GILs could transfer intact donor MHC antigen to SLT-resident cells capable of presenting the donor MHC molecules to naïve B cells. However, using a model in which GILs are photoconverted inside skin allografts, donor MHC molecules were detected on a small percentage of LN-resident leukocytes but not on photoconverted GILs homed in dLNs.

We also demonstrated in mice that allografts deliver cell-free donor MHC antigen to dSLTs via release of EVs. Graft parenchymal cells, graft endothelium, or donor passenger leukocytes retained within the graft are likely the cellular sources of donor EVs, an issue that will require further investigation. Because eukaryotic cells have been shown to respond to certain noxious stimuli by releasing EVs that promote inflammation [reviewed in (53, 54)], it is likely that surgical trauma or ischemia and reperfusion injury of the graft augment the release of donor EVs or alter its cargo. It has been previously demonstrated that graft inflammation increases donor MHC cross-dressing of recipient APCs (55).

The membranes of EVs, particularly of APC-derived exosomes, are enriched in MHC class I and class II molecules oriented with its polymorphic domains facing outward, which facilitates recognition by B cells. Unlike soluble individual donor MHC molecules, graft EVs travel without diluting their cargo of donor MHC antigen carried on the vesicle surface. After skin transplantation, graft-derived, lymph-borne small EVs were detected in the SCS of dLNs where the donor EVs were captured by SCS macrophages. The graft-derived EVs were transported across the SCS macrophages through a system of interconnected vesicles or membrane invaginations. This path resembles the mechanism of retention of vesicular stomatitis virus by SCS macrophages, and transcytosis of murine leukemia virus across SCS macrophages during transinfection of B cells under the SCS (11, 56). Exosomes share similarities with enveloped virus regarding its biogenesis, size, and entry in target cells (57) and are similarly decorated with α2,3-linked sialic acids that bind the sia-loadhesin CD169 (58).

As occurs during viral transinfection, the ability of SCS macrophages to concentrate and transport graft EVs across the SCS augments the probability that the donor EVs encounter the relatively few donor-reactive naïve B cells (about 1:100,000) in the underlying B cell follicles. We demonstrated by intravital imaging that naïve B cells recognize in a cognate fashion donor MHC antigen on EVs concentrated by SCS macrophages. Earlier studies have shown that SCS macrophages present epitopes from model antigens and pathogens to naïve B cells (9-11, 39). Our findings indicate that an analog mechanism operates in transplantation, in which presentation by SCS macrophages of donor EVs, concentrated in clusters and with each EV-bearing multiple donor MHC molecules, warrants extensive BCR cross-linking and B cell activation. Activation of alloreactive B cells depended on the physical properties of the donor vesicles and the SCS integrity, because donor EV disruption and SCS macrophage depletion both impaired alloreactive B cell activation or DSA generation.

After cognate interaction with allogeneic EVs, the primed B cells mobilized to the B cell–T cell border in seek of CD4 T cell help (59). This suggests that indirect pathway CD4 T cells are involved in B cell allosensitization against donor EVs, by recognizing donor allopeptides processed and presented by the same B cells. Allogeneic EVs are internalized and processed into allopeptides by APCs for presentation to indirect pathway CD4 T cells (19).

Donor EVs are also transferred from SCS macrophages to B cells that carry the EVs deeper into the B cell follicle. Flow cytometry analysis revealed that allogeneic EVs associate with B cells efficiently via cognate interaction and much less effectively through noncognate mechanisms, the latter likely via recognition of complement or Igs deposited on the EVs or EV ligands. As earlier studies have shown for immune complexes (9, 40), it is likely that cognate and noncognate B cells carry the donor EVs through the follicle to relay the allogeneic vesicles to FDCs to promote affinity maturation of DSAs, which will require further investigation. FDCs display endogenous EVs, different from the FDC-derived iccosomes, attached to the cell membrane (60), and here, we detected donor MHC antigen on FDCs in graft-dSLTs up to 45 days after transplantation of skin or heart allografts in mice, when donor passenger leukocytes are undetectable in graft-dSLTs. Analysis by super-resolution microscopy strongly suggested that donor MHC antigen deposited on recipient FDCs was, at least partially, delivered via EVs. However, in the absence of a methodology to specifically remove graft-derived EVs in vivo, the role of graft-derived EVs retained on recipient FDCs in persistence or affinity maturation of DSAs remains uncertain.

On the basis of our findings, pharmacological inhibition of release of graft EVs could offer a therapeutic target to prevent dissemination of donor antigen, which could minimize donor sensitization and reactivation of directly alloreactive central memory T cells and memory B cells in SLTs in presensitized recipients (61, 62). However, it is likely that more than one type of donor-derived EV is involved in B cell and T cell priming or reactivation. The family of EVs released by eukaryotic cells encompasses a broad group of vesicles with different biogenesis, compositions, and sizes released by alive or dying cells. They include exosomes (30 to 150 nm in size) generated within the endocytic compartment, microvesicles (0.1 to 1 μm in diameter) shed by the plasma membrane, multiple apoptotic cell–derived EVs, and other EVs not yet fully characterized. In the absence of reliable markers for each type of EV, the best criteria to discriminate between different donor EVs is biogenesis, which cannot be tracked back in donor EVs detected in graft-dSLTs. To our knowledge, there are no global blockers of EV biogenesis, release, or function, which could exert harmful side effects due to the multiple biological roles attributed to EVs. Among the few EV inhibitors reported so far, the neutral sphyngomyelinase inhibitor GW4869 has been shown to decrease biogenesis of small EVs, such as exosomes (63). In our hands, administration of GW4869 was associated with prolongation of cardiac allograft survival and reduction of DSA titer in serum. Because of potential off-target effects of the inhibitor, the ability of GW4869 to decrease the release of donor EVs in vivo was confirmed in cardiac allograft recipients by measuring the donor EV–mediated, donor MHC cross-dressing of recipient APCs in graft-dSLTs and their ability to activate ex vivo T cells against donor intact MHC class II molecules.

Last, our results in huMice demonstrate that our findings are not restricted to mouse models, because human grafts also release EVs that traffic and accumulate in human SCS macrophages and human donor EVs are captured by human B cells and DCs in graft-dSLTs. We ruled out potential weakness of the huMice model regarding cross-reactivity between human and mice of the primary Abs used for the immunostainings by validating that the CD169 Ab and CD63 Ab used to detect the chimeric human SCS macrophages and human graft-derived EVs, respectively, do not recognize their mouse counterparts. Our analysis by IEM of human graft-derived EVs in dLNs of huMice was qualitative and not designed to quantify expression of individual proteins on the human EVs in situ, because gold beads of different sizes contain different numbers of binding sites for the primary Ab.

Although our study analyzed the role of donor EVs in B cell allosensitization in models of acute rejection, EVs released by allografts could also participate in chronic rejection. Serum and bronchioloalveolar lavage fluid of patients with chronic rejection of lung allografts contain donor EVs with high content of donor MHC, costimulatory and adhesion molecules, lung self-antigens, and proinflammatory molecules (31, 64). Whether the EVs released by lung allografts contribute to chronic rejection via DSA elicitation or other mechanisms or are a consequence of graft tissue damage remains unknown.

One of the limitations of our study, common in the field of EVs, is the lack of models in which generation of different types of EVs can be specifically blocked in a substantial manner in vivo so the role of different types of EVs in biological functions can be assessed in situ. Besides, different types of EVs may exert similar or overlapping effects on target cells so that even depletion of one type of EVs may not have a detectable impact in vivo on the biological process analyzed. This is particularly challenging in transplantation models where donor and recipient EVs coexist in the same host. However, transplant models offer the unique situation in which different populations of EVs (donor versus recipient derived) can be tracked in situ because of expression of different MHC antigens on the vesicle surface. Our study does not exclude the contribution of other mechanisms or APCs in B cell allosensitization against donor intact HLA antigens, non-HLA antigens, and autoantigens after transplantation, as has been previously shown in other experimental models using microbial or model antigens.

In summary, our findings are consistent with a model in which allografts spread donor antigens via EVs that promote B cell allosensitization. A similar EV-mediated mechanism could also mediate reactivation of alloreactive memory B cells under the SCS (61). The recent finding that SCS macrophages restrict dissemination of tumor-derived EVs (65) suggests that SCS macrophages may facilitate or suppress traffic of EVs across the SCS under different situations, which could be exploited for therapeutic applications to restrain allosensitization. Our findings provide answers to an old enigma in transplant biology: How B cells resident in graft-dSLTs see the donor intact MHC molecules expressed by allografts. They also open possibilities for therapeutic venues for targeting donor EV biogenesis, traffic, or function to control the antidonor adaptive immune response against allografts.

MATERIALS AND METHODS

Study design

The aim of the study was to test whether B cell allosensitization and subsequent generation of de novo DSAs after transplantation are mediated by release of cell-free donor antigen via graft-derived EVs that traffic to graft-dSLTs. To this objective, we conducted skin and heart transplants in mice, in which donor passenger leukocytes are unable to migrate out of the grafts and tracked in vivo donor-derived EVs mobilized from allografts to dSLTs. Alternatively, we injected EVs labeled with a red fluorochrome, followed the vesicle traffic to dSLTs, and analyzed the interaction of the vesicles with alloreactive B cells in vivo using two-photon microscopy. We used established models of transplantation in mice, which included transplantation of wt or genetically modified nonvascularized (skin) and vascularized (heterotopic abdominal heart) allogeneic and syngeneic grafts in wt and genetically engineered recipient mice, and human skin transplantation in huMice. Mouse models of skin and heart transplantation recapitulate the immunological responses that takes place in humans. Sample size for the transplantation experiments was based on power analysis for the analysis of variance (ANOVA) test to calculate the n per group based on the significant criterion (α = 0.05), estimation of the population eta² (eta-squared) based on previous experiments run in our laboratory, and a power for ANOVA of 0.99. All groups included 10% extra animals in anticipation for potential animal death during or immediately after surgery, or after surgery complications that required euthanasia. The end point was graft rejection for graft survival experiments or predetermined PODs for imaging analysis of tissues. Animals of similar age were assigned to control and experimental groups randomly. Male or female mice were randomly selected for the experiments in accordance with the National Institutes of Health (NIH) scientific rigor policy. Mice used in experimental and control groups were housed in the same animal facility and used for the experiments by the same time to prevent batch differences. The number of animals per group for the in vivo studies and the experimental replicates for the in vitro or ex vivo studies are indicated in the figure legends. No data outliners were excluded. Assessment of graft survival between groups was performed in a blind fashion, except in those cases where it was not possible because of different strain colors of donor or recipient mice between control and experimental groups.

Single-cell suspensions of leukocytes from SLTs and graft tissues were used for mechanistic studies in vitro or ex vivo by ELISpot, PCR, flow cytometry, and ImageStream analysis. Tissue samples from grafts and SLTs were used for analysis by immunofluorescence, confocal, super-resolution, multiphoton, or IEM. Experimental and control cell and tissue samples from the same experiment were analyzed simultaneously. For analysis of multiphoton microscopy, analysis of the interaction of allogeneic EVs with alloreactive B cells was performed simultaneously with control B cells in the same movie, to prevent variations between experiments.

Mice and reagents

B6, BALB/c, C3H/HeJ (C3H), B6.SJL-Ptprc^aPepc^b/BoyJ (CD45.1 congenic), B6.Cg-Tg(Itgax-venus)1Mnz/J (CD11c-YFP), B6.C-Tg(CMV-Cre)1Cgn/J (CMV-Cre), B6.Cg-Tg(Itgax-Cre)1-1Reiz/J(CD11c-Cre), B6.129P2(C)-Ccr7^tmRfor/J (CCR7^Ko), and NOD.Cg-Prkdc^scid Il2rg^tm1Wjl Tg (CMV-Il3, CSF2, KITLG) 1Eav/MloySzJ (NSG-SGM3) mice, the later encoding human interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and stem cell factor, were purchased from the Jackson laboratory. B6 KikGR mice were generated by cross-mating B6 cytomegalovirus promoter (CMV)–Cre female mice with B6 ROSA26-loxP-stop-loxP-KikGR male mice [provided by RIKEN Brain Science Institute and generated by M. Tomura and O. Kanagawa, Kyoto University (38), with the pCAGGS expression vector licensed by J. Miyazaki, Osaka University]. MHC class II–EGFP B6 knock-in mice (generated and provided by H. Ploegh, Boston Children’s Hospital) (66), 4C B6 Rag1^−/− (Thy1.1 congenic) B6 mice (generated by T. Brennan, Duke University) (67), 3-83 Ig BALB/c mice [generated by R. Pelanda, University of Colorado Denver (41)], and Rab27a^KO B6 mice (provided by S. Catz, The Scripps Research Institute) (68) were bred in the Central Animal Facility of the University of Pittsburgh. Mice were maintained in the pathogen-free animal facility of the University of Pittsburgh School of Medicine. Animal care and handling were performed in accordance with institutional guidelines and the procedures approved by Institutional Animal Care and Use Committee (IACUC) protocol numbers 19106062, 20098112, and 20067566.

Cytokines and Abs were purchased from PeproTech, and Abs were purchased from Abcam, BD Pharmingen, BioLegend, eBioscience, Invitrogen Molecular Probes, Jackson ImmunoResearch, Thermo Fisher Scientific, Santa Cruz Biotechnology, Novus, and Cedarlane. CellTracker CM-DiI Dye, CellTracker CMAC Dye, CellTracker Carboxyfluorescein diacetate succinimidyl ester (CMFDA) Dye (CFSE), and Fluo-4 AM were from Thermo Fisher Scientific. GW4869 and methane sulfonic acid (MSA) solution were purchased from Sigma-Aldrich, and tacrolimus (FK506) was purchased from the university hospital pharmacy. The detailed list of reagents, mice, and computer software used in this study are included in the Supplementary Materials.

Generation of huMice

CD34 hematopoietic stem cells (HSCs) were collected from cord blood from placenta of healthy pregnant women following the guidelines of the Institutional Review Board (IRB) of the University of Pittsburgh. Heparinized cord blood was diluted (1:1) with phosphate-buffered saline (PBS), and peripheral blood mononuclear cells (PBMCs) were purified with two consecutive discontinuous Ficoll-Paque (1.077 g/ml; Ficoll-Paque Plus, GE Healthcare) density gradients (900g for 30 min at 18° to 20°C). Human HSCs were isolated from PBMCs by magnetic sorting with CD34 Ab microbeads (Miltenyi Biotec) and cryopreserved in liquid nitrogen until use. CD34 HSC purity by flow cytometry analysis was ≥90% with less than 1% of CD3 T cells. To reconstitute NSG-SGM3 mice, CD34 HLA-A2^Neg HSCs were thawed, washed in PBS, and cultured in RPMI 1640 and 10% fetal bovine serum (FBS) for 4 to 6 hours. A minimum of 10⁵ HSCs in 25 μl of PBS were injected in the liver of 2- to 3-day-old pups γ-irradiated (100 cGy) 6 hours prior. Percentages of human leukocyte chimerism were determined by flow cytometry analysis in peripheral blood at 12 weeks and in the spleen at the end of the experiments (14 to 16 weeks). Studies on huMice were approved by the IACUC (protocol no. 20067566) and the IRB (no. PRO13120232) of the University of Pittsburgh.

Generation of conditional transgenic mice

B6 LSL-RFP-CD63 mice were generated by the Transgenic and Gene Targeting Core of the University of Pittsburgh. A targeting vector was designed to contain the mouse CMV, followed by a loxP-flanked stop sequence [three copies of the SV40 polyadenylate poly(A) signal], TagRFP complementary DNA (cDNA) (Evrogen) fused to mouse CD63 cDNA (Sino Biological Inc.), and the phosphoglycerate kinase polyA signal. The linearized vector was micro-injected in the pronucleus of B6 zygotes, and embryos developed to the two-cell stage were transferred to the oviducts of pseudo-pregnant B6 females. Potential founders were identified in the pups by PCR genotyping of the TagRFP-mCD63 cDNA cassette (TagRFP forward primer, 5′-AACCTTCATCAACCACACCCA-3′; mCD63 reverse primer, 5′-CACCCACTGCAATGATGACCA-3′). B6 mice with ubiquitous expression of TagRFP-CD63 or under the CD11c promoter were generated by cross-mating B6 LSL-RFP-CD63 males with B6 CMV-Cre or CD11c-Cre females, respectively.

Transplantation models

For mouse skin transplants, donor mice were euthanized, and ears were removed. The ears were split with forceps into dorsal and ventral halves. The dorsal halves, which contain no cartilage, were transplanted on beds prepared on the dorsal side of recipient mice. Nebulized isofluorane and oxygen gas mixture administered via inhalatory route was used to anesthetize recipient mice. The skin on the back below the shoulder blades was shaved and cleaned with betadine and 70% ethanol. One or two skin fragments (1 cm² each) on the right and left dorsal areas between the shoulder blades and spine were sectioned and removed from its bed leaving the subdermal layer intact. One (unilateral) or two (bilateral) skin grafts were placed on the bed(s), making sure that the edges of the skin graft(s) did not overlap the surrounding skin of the recipient. Grafts were secured to the wound site with Vetbond (3M), covered with a nonadherent pad (TELFA, Tyco Healthcare) and a bandage. The bandage was removed 7 days after surgery, and graft survival was assessed by daily visual examination. Rejection was defined as the complete loss of viable tissue grafts. In some experiments, recipients were treated with FK506 via intraperitoneal injection (5 mg/kg daily).

Heterotopic (abdomen) vascularized cardiac transplantation in mice was performed with the method of Corry et al. (69). Palpation for heartbeat was conducted daily to determine organ survival. In some experiments, donors and recipients were treated with GW4869 or vehicle (DMSO-MSA). GW4869 was stored at −80°C as a 1 mM stock solution dissolved in DMSO. Right before use, the GW4869 stock solution was solubilized by addition of 5% MSA solution (5 μl of 5% MSA solution in sterile double-distilled water were added to 105 μl of GW4869 1 mM stock solution). The suspension was mixed and warmed at 37°C until clear. Each intraperitoneal injection consisted of 110 μl of the GW4869-MSA solution mixed with 200 μl of warm PBS (60 μg of GW4869 per dose) or equivalent amount of vehicle (DMSO-MSA). Donors were intraperitoneally injected with GW4869 or vehicle 5 days, 3 days, and 1 day before surgery. Recipients were treated with GW4869 or vehicle for short term (on PODs 1, 3, and 5) or long term (on PODs 1, 3, 5, and 7 and every other day thereafter).

Human skin samples were obtained from healthy individuals undergoing cosmetic plastic surgery from the Department of Pathology of the University of Pittsburgh School of Medicine, Division of University Health Science Tissue Bank, project no. 298. Human skin samples were acquired and used according to the University of Pittsburgh IRB outlines (IRB no. 0501138). Skin samples were maintained in sterile PBS at 4°C and processed 2 to 12 hours after collection. Samples were rinsed in 70% ethanol, and split-thickness explants were prepared with a skin cutaneous mini knife (Integra Padgett). The epidermal and dermal explants were cut into ~1-cm² grafts that were kept in petri dishes with RPMI 1640 and 10% FBS on ice and were transplanted within 1 to 3 hours. For skin transplantation, HLA-A2^Neg huMice were anesthetized, shaved on the back, and cleaned with betadine and 70% ethanol as aforementioned. Two skin areas (1 cm² each), on the right and left dorsal areas between the shoulder blades and spine, were cut and removed from its bed leaving the subdermal layer intact. Two HLA-A2^{Pos or Neg} human skin grafts (bilateral) were placed on the respective beds, making sure that the edges of the skin grafts did not overlap the surrounding skin of the recipient. Grafts were secured to the wound site with Vetbond and covered with a nonadherent pad and a bandage. Mice transplanted with skin or heart grafts were treated with the analgesic Buprenex for three consecutive days after surgery.

Amnis ImageStream analysis

Leukocytes from graft-dLNs (brachial) of BALB/c mice transplanted with B6 (CD45.1) or BALB/c skin grafts were Fc receptor (FcR)–blocked with CD16/CD32 Ab and incubated (30 min at 4°C) with the following Abs: phycoerythrin (PE)–H2K^b, PE-H2D^b, PerCP-Cy5-CD11c, AF647-CD45.2, and BV605-CD19. Splenocytes from BALB/c mice, naïve, or transplanted with B6 (CD45.1) hearts and treated with GW4869 or vehicle were FcR-blocked with CD16/CD32 Ab and then labeled with PE-H2K^b, PE-H2D^b, PerCP-Cy5.5-CD11c, BV605-CD19, APC-CD45.2, and APC-Cy7-CD63 Abs. Leukocytes from graft-dLNs (brachial) and mesenteric LNs of HLA-A2^Neg huMice grafted with human skin were incubated with fluorescein isothiocyanate–HLA-A2, PE-hCD45, PE-Cy5-hCD3, BV421-hCD63, APC-hCD19, and APC-Cy7-hCD11c Abs. Fluorochrome-conjugated Ab was used between 1:100 and 1:200 final concentration, diluted in PBS/1% FBS/0.1% sodium azide solution (pH 7.4 to pH 7.6). In all cases, cells were fixed in 4% paraformaldehyde-PBS immediately after labeling. Cells (2 × 10⁵ per group) were analyzed with a two-laser Amnis ImageStream analyzer at a magnification of ×60. Cell images were analyzed with the software IDEAS v6.2. Events in focus were gated by plotting the gradient root mean square (RMS) feature of the bright field of camera 1 (channel 1) against the gradient RMS feature of the bright field of camera 2 (channel 9). From the events in focus, single cells were identified as events with low area and high aspect ratio intensity (bright field of camera 1, channel 1). Single cells were selected for further analysis based on their positivity for CD45.2 (recipient leukocytes), CD11c (DCs), or CD19 (B cells).

Transmission electron microscopy

BALB/c brachial and axillary LNs draining B6 CD45.1 skin grafts (and LNs from nontransplanted controls) were removed on POD 2, pooled, cut into halves, and digested with type IV collagenase (400 U/ml) and deoxyribonuclease I (0.1 mg/ml; 20 min at 37°C). Single-cell suspensions of LNs were labeled with biotin-H2K^b and biotin-IA^b Abs and 10-nm gold-AF488-streptavidin, APC-CD11c Ab, BV605-CD19 Ab, and PE-Cy7-CD45.2 Ab (all diluted 1:200). Recipient (CD45.2) DCs (CD11c^High) and B cells (CD19) cross-dressed with donor H2K^b/IA^b (AF488^Pos) were fluorescence-activated cell sorting (FACS)–sorted and relabeled with rabbit anti-mouse CD63 polyclonal Ab (1:100) and 6-nm gold goat anti-rabbit IgG Ab (1:50). Cells were rinsed with ice-cold PBS, pelleted by centrifugation, and the pellets were fixed in cold 2.5% glutaraldehyde in 0.1 M PBS. After fixation, the pellets were rinsed in PBS, postfixed in 1% osmium tetroxide with 0.1% potassium ferricyanide for 30 min, rinsed in PBS, dehydrated through a graded series of ethanol, and embedded in epon. Semithin sections (300 nm) were cut on a Reichert-Jung microtome Ultracut, stained with 0.5% toluidine blue, and examined under the light microscope. Ultrathin sections (65 nm) were stained with uranyl acetate and Reynold’s lead citrate and examined by transmission electron microscopy.

Whole LN and spleen fragments (2 mm by 2 mm) from mice transplanted 1 or 2 days before with skin or heart allografts were fixed in 2% paraformaldehyde–0.01% glutaraldehyde–PBS (pH 7.3) (1 hour, on ice), transferred to 20% polyvinylpyrrolidone in 1.6 M sucrose buffered with 0.055 M sodium carbonate (overnight, 10°C), and then frozen to small stubs in liquid nitrogen. Semithin sections (300 nm) were cut on a Leica Ultracut 7 with a cryokit at −110°C, stained with 0.5% toluidine blue, and examined under a light microscope to determine specific tissue areas. Ultrathin sections (65 nm) collected on formvar-coated copper grids were blocked with 5% normal goat serum–PBS and treated with the Avidin/Biotin Blocking Kit (Vector Laboratories). The grids were labeled with CD169 (rat IgG2a) Ab, biotin-IA^b, biotin-H2K^b Ab, and rabbit anti-CD63 polyclonal Ab (overnight, 10°C) (all 1:100) and then incubated with 10-nm gold-streptavidin, 18-nm gold goat anti-rat IgG Ab, and 6-nm gold goat anti-rabbit IgG Ab (all diluted 1:50) for 1 hour at room temperature. Ultrathin cryosections of pellets of CD11c^High DCs, FACS-sorted from spleens of CMV-Cre X LSL-RFP-CD63 mice, were labeled with mouse anti-RFP Ab and rabbit anti-CD63 polyclonal Ab (1:100) overnight at 10°C, followed by 10-nm gold anti-mouse Ig Ab and 6-nm gold goat anti-rabbit IgG Ab (1:50) for 1 hour at room temperature.

Graft-dLNs of HLA-A2^Neg huMice transplanted with HLA-A2^{Pos or Neg} skin grafts were removed on POD 3 and processed for IEM as aforementioned. Ultrathin cryosections (65 nm) on formvar-coated copper grids were blocked with 5% normal goat serum in PBS and treated with the Avidin/Biotin Blocking Kit (Vector Laboratories). The grids were incubated overnight at 10°C with purified mouse anti-human CD169 Ab, biotin–HLA-A2 Ab, and anti-human CD63 rabbit monoclonal Ab (all 1:100). Grids were then labeled with 18-nm gold anti-mouse IgG, 10-nm gold-streptavidin, and 6-nm gold goat anti-rabbit IgG (all 1:50) for 1 hour at room temperature.

After postembedding immunogold staining, grids were rinsed in PBS, and sections were fixed in 2.5% glutaraldehyde and then rinsed in PBS and distilled water. The rinsed sections were counterstained with 2% neutral uranyl acetate and 4% uranyl acetate and then coated with methyl cellulose. All specimens were analyzed on a JEOL 1400 transmission electron microscope with a side-mount AMT 2k digital camera. The size of the EVs on transmission electron images was measured with the MetaMorph Offline 7.7.50 software.

Cell tracking by photoconversion

B6 KikGR mice were generated by cross-mating B6 ROSA26-loxP-stop-loxP-KikGR males with B6 CMV-Cre females. Only B6 KikGR mice (CMV-floxed) with ≥98% expression of KikGR-green assessed by flow cytometry in PBMCs were used as graft recipients. KikGR B6 mice transplanted on the dorsal side with BALB/c skin grafts were anesthetized and protected with a cover of aluminum foil with a perforation exposing only the grafts. The skin allografts were left untreated (control) or exposed to violet light (25 μW/cm², 30 s + 30 s, 1 min apart) every day, using an optogenetics light-emitting diode violet lamp (Prizmatix). On POD 3 or 7, the skin allografts and graft-dLNs (brachial and axillary) were removed. The allografts were cut into small fragments, digested in RPMI 1640 medium containing Liberase (0.2 mg/ml; Roche) and DNase I (0.1 mg/ml; 60 min at 37°C), and mixed repeatedly with a pipette with a blue tip with the tip cut. Single cells were passed through 70-μm cell strainers, and digestion was stopped with ice-cold 10 mM EDTA-PBS. Single-cell suspensions of skin allografts were labeled with APC-CD45.2 Ab, and the percentage of recipient leukocytes (KikGR-green) photoconverted into KikGR-red was determined by flow cytometry analysis. Single-cell suspensions of graft-dLNs were incubated with CD16/CD32 Ab (to block FcR) and then labeled (30 min at 4°C) with PE-Cy7-CD45.2 Ab, APC-H2K^d Ab, APC-IA^d Ab, and eFluor780 Fixable Viability Dye. Fluorochrome-conjugated Ab was used between 1:100 and 1:200 final concentration, diluted in PBS/1% FBS/0.1% sodium azide solution (pH 7.4 to pH 7.6). The skin allograft and graft-dLN cells were analyzed by flow cytometry immediately after labeling, without cell fixation. To calculate absolute number of leukocytes, 50 μl of CountBright absolute counting beads were added to each FACS tube immediately before analysis.

Generation and purification of EVs

EVs were purified from culture supernatants of DCs following the guidelines of the International Society of Extracellular Vesicles (70). Mouse DCs were generated from BM precursor cells cultured in RPMI 1640 supplemented with 10% FBS, l-glutamine (2 mM), nonessential amino acids (1×), sodium pyruvate (0.11 mg/ml), Hepes (10 nM), tissue culture grade 2-mercaptoethanol (0.55 mM), penicillin (100 U/ml), streptomycin (100 μg/ml), GM-CSF (1000 U/ml), and IL-4 (500 U/ml), as previously described (19). DCs were maintained in medium with EV-free FBS during the last 48 hours of culture (DC viability of ≥95% by flow cytometry based on exclusion of Fixable Viability Dye). EV-free FBS was prepared by 100,000g centrifugation (18 hours at 4°C) of FBS prediluted 1:4 in complete RPMI 1640 culture medium without the cytokines, followed by filtration through 0.22-μm pore filters. DC culture supernatants (day 6) were centrifuged at 2000g (20 min) and 10,000g (30 min), both at 4°C. The 10,000g supernatants were concentrated by ultrafiltration on sterile Vivacell 100 (Sartorius) filters (2800g for 35 min at 4°C). The filtered supernatants were adjusted to a volume of 11 ml with PBS and centrifuged at 110,000g for 90 min at 4°C. The 110,000g pellets with the EVs were incubated with CM-DiI–PBS (0.1 μg/μl) (300 μl final volume) at 37°C for 5 min and then on ice for 20 min. The CM-DiI–labeled EVs were further purified and isolated from the free dye using iodixanol (OptiPrep) gradients, as previously described (71), with minor modifications. Briefly, 300 μl of CM-DiI–labeled EVs were mixed with 6 ml of 30% (w/v) OptiPrep solution in sucrose buffer [0.25 M sucrose, 10 mM tris (pH 8.0), and 1 mM EDTA (pH 7.4)], allowed 30-min mixing on a rocker to equilibrate the solution, and then transferred into Ultra-Clear (Beckman Coulter) sterile 11-ml tubes. Next, 3 ml of 20% (w/v) OptiPrep solution in sucrose buffer, followed by 2.5 ml of 10% (w/v) OptiPrep solution in sucrose buffer, was successively layered on top on the 30% OptiPrep solution containing the EVs. The tubes with the OptiPrep gradients were centrifuged at 110,000g (16 hours at 4°C). The fractions containing the EVs were harvested, washed in PBS, and centrifuged at 110,000g for 90 min, and the EV pellets were collected in 200 μl of PBS. For some experiments, the EVs were left unlabeled and untreated or disrupted with five freeze-thaw cycles in liquid nitrogen, followed by treatment with the detergent-free exosomal protein extraction kit (101Bio). Next, the disrupted EVs were loaded in Slide-A-Lyzer MINI Dialysis Devices (10K molecular weight cutoff, 0.5 ml; Thermo Fisher Scientific) and dialyzed twice against PBS (pH 7.6), first for 2 hours at room temperature and then overnight at 4°C.

Immunoisolation of B6 DC-derived small EVs was performed with streptavidin-Dynabeads (4.5 μm) preincubated with biotin-mouse CD63 Ab (clone NVG-2), biotin-mouse CD9 Ab (clone MZ3) and biotin-mouse CD81 Ab (clone TAPA-1), or biotin-irrelevant rat IgG2a (clone RTK2758), at a ratio 4 μg of biotin-Ab per 10⁷ beads (60 min at room temperature). Beads were washed with PBS and 0.1% bovine serum albumin (BSA) filtered through a 0.2-μm filter, and 25-μg total protein of EVs in 100 μl of PBS and 0.1% BSA was incubated with CD63 Ab-Dynabeads or irrelevant IgG-Dynabeads (20 μl of beads per sample, from batches of 10⁷ beads/ml), in 2-ml Sarstedt tubes, overnight at 4°C, on a sample mixer. Next, tubes were centrifuged to collect the bead-bound EVs in the pellets, and the supernatants (100-μl final volume) were incubated with CD9/CD81 Ab-Dynabeads or irrelevant IgG-Dynabeads (20 μl of beads per sample, from batches of 10⁷ beads/ml). After EV incubation, Dynabeads were washed with PBS and 0.1% BSA, transferred to FACS tubes, and labeled with AF488-irrelevant IgG, AF488-H2K^b Ab, and AF488-IA^b Ab, PerCP/Cy5.5 irrelevant IgG, or PerCP/Cy5.5-CD63 Ab (all Ab 1:200) for 60 min at 4°C on a sampler mixer. Beads were analyzed by flow cytometry, and only bead singlets were included in the results.

For isolation of EVs from mouse serum, 200 μl of serum was centrifuged at 1500g for 10 min, followed by 10,000g for 10 min. The EVs contained in 150 μl of supernatants were purified with qEVsingle (70 nm) size exclusion columns (Izon Science), according to the manufacturer’s protocol.

The amount of protein in the EV preparations was assessed with a NanoDrop 2000c, and the morphology of the EVs was analyzed by transmission electron microscopy. The average size of the EVs was measured with a LM10 NanoSight’s instrument equipped with a high-sensitivity electron-multiplying charge-coupled device camera and the NTA 2.0 software (NanoSight), as previously described (72). Samples were measured by NTA at room temperature, at concentrations ranging from 1.09 × 10⁸ to 10.68 × 10⁸ particles/ml after 1:100 to 1:1000 dilutions in batches of PBS BioPerformance certified for molecular biology (MilliporeSigma) that were previously validated in our LM10 NanoSight’s instrument to be particle-free. Samples were run at 12.5 frames/s, with five repeats of 60 s each, within a range of 20 to 60 particles per frame, with more than 350 completed tracks per video, camera shutter speed at 65 ms, pump speed set at 50, and blur and minimum track length set at automatic. NTA parameters were kept identical throughout the whole capture and analysis when comparing different samples. Results were analyzed with the NTA 2.3 software with default settings.

Analysis by two-photon microscopy

Two-photon microscopy was performed intravital on pLNs, on explanted pLNs fixed with 2% paraformaldehyde-PBS (10 min, on ice), or on living explanted graft-dLNs. For intravital microscopy, mice were anesthetized with a nebulized isofluorane and oxygen gas mixture and immobilized on a custom-built platform kept at 37°C on a heating block. Mouse body temperature was monitored with a temperature probe and maintained at 37°C during imaging. For intravital imaging, the right pLN was surgically exposed, embedded in 2% low-melting agarose, and covered with a coverslip. For ex vivo imaging, pLNs were removed, fixed in 2% paraformaldehyde-PBS, glued on a petri dish, and immersed in PBS.

To visualize capture of EVs in vivo, CM-DiI–labeled EVs (~55 × 10⁸ particles per injection) were subcutaneously injected in the footpad of BALB/c mice after LN surgery, and the arrival of the labeled EVs to pLNs was recorded immediately. For in vivo labeling of SCS macrophages in pLNs, 0.75 μg of AF488- or AF647-conjugated Ab against CD169 (clone 3D6.112, BioLegend) was injected in the footpad 16 hours before imaging. To visualize B cell interactions with EVs, CMAC-labeled 3-83 Ig B cells and CFSE-labeled wt BALB/c B cells (10⁷ of each per mouse) were intravenously injected in BALB/c mice 16 hours before imaging, and CM-DiI–labeled allogeneic (B6) or syngeneic EVs were injected in the footpad 2 hours before imaging. To identify calcium flux in alloreactive B cells, CMAC-labeled 3-83 Ig B cells loaded with Fluo-4 AM (10⁷ cells per mouse) were intravenously injected in BALB/c mice 16 hours before imaging, and CM-DiI–labeled allogeneic (B6) or syngeneic EVs were injected in the footpad 2 hours before imaging.

To image the interaction of B cells with SCS macrophages in explanted graft-dLNs alive, BALB/c mice were transplanted with B6 skin allografts on the dorsal side. The following day, the recipient mice were injected intravenously with CellTrace Violet–labeled 3-83 Ig B cells and CellTrace Far Red–labeled wt BALB/c B cells (10⁷ of each per mouse) together with 0.75 μg of AF488-CD169 Ab, the latter administered subcutaneously under the skin allograft. After 16 to 18 hours, the graft-dLNs (brachial) were removed under anesthesia and immediately glued to a 60-mm petri dish with Vetbond (3M) and covered with prewarmed (37°C) phenol red-free RPMI 1640 medium supplemented with 2% FBS exposed overnight to an atmosphere of 5% CO₂/18.6% O₂/70.2% N₂ in a tissue culture incubator. The petri dish containing the explanted LN in culture medium was placed on a heating block in the image chamber, and the entire system was maintained at 37°C during imaging.

Images were acquired with an Olympus FluoView FV1000 microscope (Olympus America, Center Valley, PA) equipped with a Mai Tai DeepSee laser (Spectra-Physics), an A1R MP Nikon multiphoton system (Nikon Instruments Inc.) equipped with a Chameleon infrared (IR) laser (Coherent Inc.), a Prior ProScan III stage (Prior Scientific Inc.), and an MCL Nano-Drive objective Z-drive (Mad City Labs Inc.), or a custom-built Leica SP8 DIVE microscope equipped with six HyD detector units, resonance scanner, and two Spectra-Physics femtosecond-pulsed lasers with two tunable and one fixed laser line suitable for two-photon excitation.

Lasers were tuned and mode-locked to 760 to 780 nm for excitation of CM-Dil, AF488, CFSE, Fluo-4 AM, CMAC, CellTrace Violet, CellTrace Far Red, and TagRFP and to 870 nm for excitation of YFP, AF647, and secondary harmonic signal. Microscopes were equipped with four nondescanned photomultiplier tubes. The following sets of filters were used: (i) 420- to 460-nm (CMAC and secondary harmonic), 495- to 540-nm (CFSE, AF488, YFP, and Fluo-4 AM), 573- to 647-nm (CM-Dil), and 660- to 760-nm (AF647) detection filters (Olympus) and a 690-nm IR cutoff filter (Olympus), in the Olympus FV1000 microscope; (ii) 460- to 500-nm (CMAC and secondary harmonic), 500- to 550-nm (CFSE, AF488, YFP, and Fluo-4 AM), 570- to 620-nm (CM-Dil), and 665- to 760-nm (AF647) detection filters (Nikon) and a 750-nm IR cutoff filter (Nikon), in the A1R MP Nikon multiphoton system; or (iii) 555-nm primary dichroic mirror with 495- and 640-nm secondary dichroic mirrors with band-pass filters 460/60 nm (CellTrace Violet), 520/35 nm (AF488), 575/19 nm (CM-Dil, tagRFP), and 655/15 nm (CellTrace Far Red) in the Leica multiphoton system. Image acquisition was performed with a water immersion objective (×25; numerical aperture, 1.05). Stacks of 12 optical sections with 4-μm Z-spacing were acquired every 30 s for 30 to 40 min with the Olympus FV1000 microscope. Stacks of 33 optical sections with 3-μm Z-spacing were acquired every 45 s for 30 to 40 min with the A1R MP Nikon multiphoton microscope and the Leica SP8 DIVE microscope. The resolution was 512 pixels by 512 pixels. Brightness and laser power were adjusted on the basis of the imaging depth and kept below phototoxic intensities. Images were acquired with the NIS-Elements v5.11.01 software (Nikon Instruments Inc.), FLUOVIEW FV31S-SW Viewer software (Olympus), or the Leica Application Suite X (Las X) 3.5.519976 (Leica) and then analyzed using Imaris x64 version 8 software (Bitplane). Background subtraction was performed on all channels equally. Videos were edited with Adobe Premiere Pro CC 2017 version 11.0 software.

Statistical analysis

GraphPad Prism was used for statistical analyses. Results are expressed as means ± SD. Multiple comparisons on a single dataset were performed by one-way ANOVA, followed by Tukey-Kramer multiple comparisons test. Comparisons between two groups were performed with two-tailed Student’s t test. For comparison of two nonparametric datasets, the Mann-Whitney U test was used. Graft survival was compared by Kaplan-Meier analysis and the log-rank test. In all experiments, P ≤ 0.05 was considered significant.

Supplementary Material

Data file S1. Raw data

Data file S1. Raw data.

NIHMS1786222-supplement-Data_file_S1__Raw_data.xlsx^{(31.9KB, xlsx)}

Zeng etat Sup Mat STM 2022

Fig. S1. Migration to the spleen of donor DCs from skin allografts.

Fig. S2. Effect of CCR7 deletion on content of passenger leukocytes in allografts and homing of allogeneic DCs to dLNs.

Fig. S3. Mobilization of passenger leukocytes out of wt and CCR7^KO allografts.

Fig. S4. Cell-free donor MHC antigen is trapped by graft-dLNs.

Fig. S5. SCS macrophages of dLNs internalize donor-derived EVs released by skin allografts.

Fig. S6. Acquisition of cell-free donor alloantigen by recipient cells in the spleen after cardiac transplantation.

Fig. S7. MHC molecules are carried by different subsets of small EVs.

Fig. S8. Skin allografts release donor antigen via small EVs.

Fig. S9. Alloreactive B cells recognize EVs in an allospecific fashion.

Fig. S10. Quantification of depletion of SCS macrophages in pLNs by clodronate liposomes.

Fig. S11. Donor intact MHC molecules and passenger cells in graft-dSLTs on POD 45.

Fig. S12. Effects of GW4869 on adaptive immunity.

Fig. S13. Effect of GW4869 treatment or deletion of donor Rab27a on cardiac allograft rejection.

Fig. S14. Model for detection of traffic of human donor cell-free antigen to human SCS macrophages in graft-dLNs.

Fig. S15. Detection specificity of human SCS macrophages loaded with donor HLA-A2 antigen in huMice LNs draining human skin grafts.

Fig. S16. Assessment of multiplexed IEM and species cross-reactivity of anti-human CD63 Abs.

Table S1. Reagents and Resources.

NIHMS1786222-supplement-Zeng_etat_Sup_Mat_STM_2022.pdf^{(3.3MB, pdf)}

Movie S1

Movie S1. SCS macrophages of graft-dLNs trap cell-free RFP released by RFP-CD63 skin allografts.

Download video file^{(36.1MB, mp4)}

Movie S2

Movie S2. Uptake of allogeneic EVs by macrophages and DCs in dLNs.

Download video file^{(33.4MB, mp4)}

Movie S3

Movie S3. Traffic of allogeneic EVs through SCS macrophages in dLNs.

Download video file^{(22.2MB, mp4)}

Movie S4

Movie S4. Interaction between alloreactive B cells and SCS macrophages loaded with allogeneic EVs.

Download video file^{(10.2MB, mp4)}

Movie S5

Movie S5. Alloreactive B cells make prolonged contacts with SCS macrophages carrying allogeneic EVs.

Download video file^{(24.6MB, mp4)}

Movie S6

Movie S6. Alloreactive B cells make stable interactions with SCS macrophages of LNs draining allografts.

Download video file^{(24.8MB, mp4)}

Movie S7

Movie S7. Steady contacts of alloreactive B cells with SCS macrophages carrying EVs released by donor DCs of skin allografts.

Download video file^{(36.4MB, mp4)}

Movie S8

Movie S8. Calcium flux in alloreactive B cells after interaction with SCS macrophages loaded with EVs.

Download video file^{(24MB, mp4)}

Movie S9

Movie S9. Alloreactive B cells transport allogeneic EVs captured from SCS macrophages.

Download video file^{(28.1MB, mp4)}

Acknowledgments:

We thank C. Wallace and G. Gibson for the technical support in two-photon microscopy.

Funding:

This work was supported by grants from the NIH R01 HL130191 (to A.E.M.), R01 AI148690 (to A.E.M.), R01 AR068249 (to A.T.L.), R01 AR071277 (to A.T.L.), R01 AI052310 (to R.P.), U01 AI132758 (to G.C.), R01 AI145881 (to M.H.O), S10OD025041 (to S.C.W.), and S10OD016236 (to S.C.W.). This work was benefitted from the ImageStreamX Mark II grant NIH 1S10OD019942-01 and the NIH Shared Instrument grant no. 1S10OD021627-01.

Footnotes

SUPPLEMENTARY MATERIALS

stm.sciencemag.org/cgi/content/full/13/585/eabb0122/DC1

Competing interests: The authors declare that they have no competing interests.

Data and materials availability: All data associated with this study are in the paper or the Supplementary Materials. Materials and animals created in this study will be available for the scientific community by contacting the corresponding author and completion of a material transfer agreement.

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