Donor dendritic cell-derived exosomes promote allograft-targeting immune response

Abstract

The immune response against transplanted allografts is one of the most potent reactions mounted by the immune system. The acute rejection response has been attributed to donor dendritic cells (DCs), which migrate to recipient lymphoid tissues and directly activate alloreactive T cells against donor MHC molecules. Here, using a murine heart transplant model, we determined that only a small number of donor DCs reach lymphoid tissues and investigated how this limited population of donor DCs efficiently initiates the alloreactive T cell response that causes acute rejection. In our mouse model, efficient passage of donor MHC molecules to recipient conventional DCs (cDCs) was dependent on the transfer of extracellular vesicles (EVs) from donor DCs that migrated from the graft to lymphoid tissues. These EVs shared characteristics with exosomes and were internalized or remained attached to the recipient cDCs. Recipient cDCs that acquired exosomes became activated and triggered full activation of alloreactive T cells. Depletion of recipient cDCs after cardiac transplantation drastically decreased presentation of donor MHC molecules to directly alloreactive T cells and delayed graft rejection in mice. These findings support a key role for transfer of donor EVs in the generation of allograft-targeting immune responses and suggest that interrupting this process has potential to dampen the immune response to allografts.

Figure 1. Transfer of donor MHC antigen in graft-draining lymphoid organs.

(A) Migrating donor (BALB/c) DCs (IA^d+) in T cell areas of the recipient (B6, H2^b) spleen (arrows, insets). Confocal microscopy, original magnification, ×200. BALB/c cDCs (CD45.2⁺CD45.1^–CD11c⁺) homed in the recipient (B6) spleen were undetectable by flow cytometry. Numbers indicate percentages of cells in the corresponding quadrant. Dot plots are representative of 1 untreated or recipient mouse of 3 per time point. (B) Quantities of donor cells mobilized from BALB/c heart grafts to the recipient (B6) spleens estimated by genomic PCR. Mean ± SD, 3 mice per variable. ND, not detected. (C) A donor DC (IA^{d hi}, arrow) in the recipient spleen next to DCs expressing IA^{d dim} or the IA^b (B6)–IEα_52–68 (BALB/c) complex (arrowheads, detected with the Yae Ab) that likely corresponded to recipient DCs that acquired donor IA^d or donor IEα_52–68 peptide, respectively. Confocal microscopy, original magnification, ×200. (D) FACS analysis of numbers of recipient (B6) splenic APCs with donor H2K^d or IA^d molecules at successive PODs after transplantation of BALB/c hearts. Mean ± SD, 3 mice per variable. P values were generated by 1-way ANOVA followed by Tukey-Kramer multiple comparisons test.

Figure 2. Recipient cDCs acquire donor MHC molecules through EVs.

(A) FACS sorting of recipient (B6) cDCs bearing donor H2K^d, analyzed 3 days after transplantation of BALB/c hearts. (B) Transmission EM (TEM) image of a recipient cDC, sorted in R1 of A, showing EVs carrying donor H2D^d and IA^d. The area in the small rectangle is shown at higher magnification on the right. Recipient cDCs did not express donor (BALB/c) MHC directly on the surface — a section of the plasma membrane is shown at higher magnification below, original magnification, ×2,500–×10,000. One image representative of 60 cells analyzed with gold-labeled EVs attached. Dot plot: size of EVs attached to recipient cDCs. (C) Recipient cDC, FACS-sorted in R1 of A, and labeled with biotin-irrelevant Ab, as a control. Bars: number of 5-nm gold-positive EVs on recipient cDCs, FACS-sorted in R1 of A, and labeled with biotin-H2D^d-IA^d or control biotin-irrelevant Abs. (D) Cluster of EVs, attached to a recipient (B6) cDC, FACS-sorted in R1 of A, expressing donor H2D^d and IA^d, and CD9 or CD63. One image representative of 50 cells analyzed with gold-labeled EVs attached.

Figure 3. Recipient cDCs that acquire intact donor MHC molecules in vivo promote T cell immunity.

(A) Recipient cDCs carrying donor MHC (FACS-sorted in R1 of Figure 2A) triggered proliferation of 2C CD8 T cells against BALB/c H2L^d. (B and C) Expression of T cell activation/effector markers (B) and cytokine secretion (C) of 2C T cells following stimulation by recipient (B6) splenic cDCs carrying donor MHC (FACS-sorted in R1 of Figure 2A), or by control cDCs. Numbers in dot plots indicate percentages of cells in the corresponding quadrants. IL-5 and IL-17A were undetectable by ELISA. In A–C, 1 representative of 4 experiments is shown. P values were generated by 1-way ANOVA followed by Tukey-Kramer multiple comparisons test.

Figure 4. Passage of MHC molecules via transfer of exosomes between DCs in vitro.

(A) Passage of IA^d from CFSE-labeled BALB/c cDCs to B6 cDCs in vitro (analyzed by FACS). Images of cDCs, FACS-sorted in R1, showing (BALB/c) H2D^d and IA^d transferred to (B6) cDCs through EVs expressing CD9 and CD63. R2: BALB/c (donor) cDCs releasing exosomes bearing H2D^d and IA^d (rectangle). R3: Recipient cDCs do not express donor MHC on their surface. Results are representative of 3 experiments. Transmission EM, original magnification, ×20,000–×60,000. Images are representative cells from 4 independent experiments. (B) Size of EVs bearing BALB/c H2D^d and IA^d, and attached to B6 cDCs. (C) Images of EVs expressing BALB/c H2D^d and IA^d, and CD9, which have been internalized by (B6) cDCs. Recipient cDCs were FACS-sorted in R1 (A), then labeled with biotin-H2K^d and -IA^d Abs plus CD9 Ab, followed by gold-conjugated secondary reagents, and then maintained at 37°C (30 minutes) to promote internalization of the EVs. Original magnification, ×20,000–×80,000. A representative single cell from 1 of 2 independent experiments is shown.

Figure 5. Efficiency of transfer of intact MHC molecules via exosomes between DCs.

(A) Transfer efficiency of H2K^d and IA^d from BALB/c to B6 cDCs (analyzed by FACS). Mean ± SD. One representative experiment of 2. (B) Percentages of B6 cDCs that acquired H2K^d and IA^d from BALB/c cDCs following culture with BALB/c cDCs together, or separated by 0.4-μm-pore transwells (analyzed by FACS). Results are representative of 4 experiments. Numbers in dot plots indicate percentages of cells in the corresponding quadrant. (C) Effect of imipramine or DEVD added to the culture medium, or pretreatment of donor BALB/c DCs with Rab27a siRNA, on transfer of IA^d from BALB/c DCs to B6 cDCs in vitro (analyzed by FACS). Results from 1 representative of 4 independent experiments are shown. P values were generated by 1-way ANOVA followed by Tukey-Kramer multiple comparisons test.

Figure 6. Recipient cDCs acquire clusters of donor-derived exosomes in vivo.

(A) Time-lapse analysis (confocal) of transfer of RFP-tagged exosome clusters from a migrating CD63-RFP BALB/c DC (blue nucleus + RFP-exosomes) to a lymph node cDC (YFP⁺, in green). Scale bar: 1 μm. (B) Side view of A. The arrow indicates internalized RFP-tagged EVs. (C) Left: 3D panoramic view (confocal) of a lymph node of a CD11c-YFP B6 mouse injected with CD63-RFP BALB/c DCs. Right: RFP-exosome clusters in relationship to host YFP⁺ cDCs, on the same image analyzed with Imaris X64. Red dots: RFP-exosomes on YFP⁺ B6 cDCs. Yellow dots: RFP-exosomes internalized by YFP⁺ B6 cDCs. Blue dots: RFP-exosomes in CD63-RFP BALB/c DCs, free or captured by YFP^– cells. (D) 3D view (multiphoton) of the spleen of a CD11c-YFP B6 mouse injected i.v. with CD63-RFP BALB/c DCs. Arrow: interaction between injected CD63-RFP BALB/c DCs and host YFP⁺ cDCs. Scale bar: 15 μm. (E) Analysis (Imaris X64) of transfer (arrows) of RFP-exosome clusters from a CD63-RFP BALB/c DC to host YFP⁺ cDCs (in green) in the spleen. In A–E, results are representative of 4 independent experiments.

Figure 7. Transfer of exosomes promotes cDC maturation in the spleen.

(A) Recipient MHC class II (B6, IA^b) and CD86 expression by cDCs and plasmacytoid DCs (pDCs) from spleens of CD45.1 B6 mice transplanted with CD45.2 BALB/c hearts, analyzed by FACS on successive PODs. Results are representative of 3 mice per variable. (B) Effect of transfer of RFP-tagged exosomes between migrating CD63-RFP BALB/c BMDCs injected i.v. and spleen-resident cDCs of CD11c-YFP B6 mice. Numbers in dot plots indicate percentages of cells in the corresponding quadrants. CD63-RFP BALB/c BMDCs were matured by overnight incubation with IL-1β plus TNF-α. Comparison by FACS analysis of expression of endogenous (B6) MHC class II (IA^b), CD40, CD80, CD86, and PD-L1 between YFP⁺ cDCs without and with RFP⁺ content, analyzed 16 hours after BMDC injection. Results are from representative experiments with 4 mice per group.

Figure 8. Effect of different EVs released by DCs on DC maturation.

(A) EM analysis of MVs and exosomes secreted by BALB/c BMDCs matured by incubation with IL-1β and TNF-α. Original magnification, ×20,000. (B) Western blot analysis of the endoplasmic reticulum protein gp96 (control), the exosome-associated protein CD81, and the DC activation/maturation marker CD86 on different EVs released by BALB/c BMDCs. One representative Western blot of 2 is shown. (C) Detection of donor (BALB/c) MHC molecules (H2D^d-IA^d) and CD86 on exosomes (arrow) from BALB/c mature BMDCs transferred to an acceptor B6 BMDC (asterisk). Transmission EM, original magnification, ×20,000. (D) Expression of endogenous MHC class II (IA^b) (analyzed by FACS) on the surface of B6 BMDCs untreated or incubated with MVs or exosomes from BALB/c BMDCs. As a negative control, exosomes were added after 5 freeze/thaw cycles (F/T). Results are from 1 representative experiment of 3. P values were generated by 1-way ANOVA followed by Tukey-Kramer multiple comparisons test.

Figure 9. Transfer of exosomes released by mature DCs promotes maturation of the acceptor DCs.

(A) Expression of endogenous MHC class II (IA^b), CD40, CD80, CD86, and PD-L1 (by FACS) on B6 BMDCs untreated or incubated with MVs or exosomes from BALB/c mature BMDCs. One representative of 3 experiments is shown. (B) Ability of B6 BMDCs, untreated or exposed to MVs or exosomes from mature BALB/c BMDCs, to promote proliferation of naive (third-party) C3H T cells in CFSE–mixed leukocyte cultures analyzed by FACS. Numbers in dot plots indicate percentages of cells in the corresponding quadrants. In A and B, 1 representative experiment of 4 is shown.

Figure 10. Recipient cDCs present donor MHC molecules to directly alloreactive T cells after heart transplantation.

(A) Survival of BALB/c cardiac grafts in CD11c-DTR-B6 BM chimeras depleted of recipient cDCs. Recipient numbers are in parentheses. (B) Quantification by immunofluorescence microscopy of donor (BALB/c) cDCs (CD11c⁺IA^d+) on tissue sections of spleens of B6 (H2^b) recipients, on successive PODs. Results represent the analysis of 10 panoramic sections of each spleen per POD and animal group. Results were analyzed with 1-way ANOVA followed by Tukey-Kramer multiple comparisons test. Cells were counted with MetaMorph Offline 7.7.50 software. NS, not significant; ND, not detected. (C) Top: Donor (BALB/c) cDCs detected on tissue sections of spleens from DT-treated WT B6 BM chimeras (control) and DT-injected CD11c-DTR BM chimeras were identified by expression of IA^{d hi} (green) and CD11c (red). Bottom: Homing of donor (BALB/c, IA^d+) cDCs (green) to splenic T cell areas (red) in DT-treated WT B6 BM chimeras (control) and DT-injected CD11c-DTR BM chimeras. Arrows indicate the donor DCs shown in detail in the insets. Nuclei were stained blue with DAPI. Immunofluorescence microscopy, original magnification, ×400. Sections are representative of 3 animals per variable. (D) Enzyme-linked ImmunoSpot (ELISPOT) analysis of the recipient T cell response against donor MHC molecules (direct pathway) or donor-derived peptides presented by recipient MHC molecules (indirect pathway) in the spleen on POD 7. Results were pooled from 3–4 mice per group. P values were generated by 1-way ANOVA followed by Tukey-Kramer multiple comparisons test. (E) Anti-donor (BALB/c) Ab titers in serum on POD 7. Recipient numbers are in parentheses.

Figure 11. Transfer of donor MHC molecules after skin transplantation.

(A) Top dot plots: Detection by FACS of donor (CD45.2⁺CD45.1^–) cells migrated from BALB/c (CD45.2⁺) fully mismatched skin allografts in B6 (CD45.1⁺) mice. Donor cells were undetectable by FACS within the “live cell gate” on PODs 1, 3, and 7, in the spleen (A) and in the draining lymph nodes (axillary + inguinal, not shown). Bottom dot plots: Detection by FACS of donor MHC class I (H2K^d + H2D^d) molecules on recipient (CD45.1⁺CD45.2^–) splenic cDCs. Recipient splenic CD8α⁺ and CD8α^– cDCs acquired donor MHC class I molecules. Numbers in dot plots indicate percentages of cells in the corresponding quadrants. Results are representative of 6 mice per time point. (B) Left: FACS analysis of intensity of expression of donor MHC class I (H2K^d + H2D^d) and class II (IA^d) molecules on recipient (B6) splenic cDCs after BALB/c skin transplantation. Right: Percentages of recipient (B6) splenic cDCs cross-dressed with donor MHC class I (H2K^d + H2D^d) and class II (IA^d) molecules, analyzed by FACS after BALB/c skin transplantation. Results were pooled from 2 experiments, each with 3 mice per time point. P values were generated by 1-way ANOVA followed by Tukey-Kramer multiple comparisons test. (C) Analysis by ImageStream technology of recipient (B6, CD45.1⁺) splenic cDCs (CD11c⁺) cross-dressed with donor (BALB/c) MHC class I (H2K^d + H2D^d) and class II (IA^d) molecules, both located in spots containing the exosome marker CD63. ImageStream, original magnification, ×60, 5,000 cells analyzed.