Extracellular vesicles derived from injured vascular tissue promote the formation of tertiary lymphoid structures in vascular allografts

Abstract

Tertiary lymphoid structures (TLS) accumulate at sites of chronic injury where they function as an ectopic germinal center, fostering local autoimmune responses. Vascular injury leads to the release of endothelial-derived apoptotic exosome-like vesicles (ApoExo) that contribute to rejection in transplanted organs. The purpose of the study was to evaluate the impact of ApoExo on TLS formation in a model of vascular allograft rejection. Mice transplanted with an allogeneic aortic transplant were injected with ApoExo. The formation of TLS was significantly increased by ApoExo injection along with vascular remodeling and increased levels of antinuclear antibodies and anti-perlecan/LG3 autoantibodies. ApoExo also enhanced allograft infiltration by γδT17 cells. Recipients deficient in γδT cells showed reduced TLS formation and lower autoantibodies levels following ApoExo injection. ApoExo are characterized by proteasome activity, which can be blocked by bortezomib. Bortezomib treated ApoExo reduced the recruitment of γδT17 cells to the allograft, lowered TLS formation, and reduced autoantibody production. This study identifies vascular injury-derived extracellular vesicles (ApoExo), as initiators of TLS formation and demonstrates the pivotal role of γδT17 in coordinating TLS formation and autoantibody production. Finally, our results suggest proteasome inhibition with bortezomib as a potential option for controlling TLS formation in rejected allografts.

Keywords: antigen presentation/ recognition; autoantibody; basic (laboratory) research/science; cell death: apoptosis; immunobiology; rejection: vascular; vasculopathy.

Figure 1

Apoptotic exosome‐like vesicles (ApoExo) are novel triggers of IL‐17 production and tertiary lymphoid structure (TLS) formation in vascular allografts. Mice were injected with the vehicle (n = 13) or ApoExo (n = 16) for 3 weeks posttransplantation: (A) Aortic allograft sections stained with H&E, CD20, CD3, AID, and IL‐17 (magnification: 5×; magnification of right inset panels: 20×). (B) Ratio intima/media in the allografts. (C) Mean number of TLS per allograft. Neointima‐media and perivascular quantification of CD20⁺ B cells (D), CD3⁺ T cells (E), AID (F), and IL‐17 (G) staining in each high‐power field of the allografts. Data were pooled from 3 independent experiments and expressed as means ± SEM. Comparison with the vehicle was done with a Student's t test

Figure 2

Apoptotic exosome‐like vesicles (ApoExo) trigger the production of anti‐LG3 IgG and ANA in allografted mice. Anti‐LG3 (A), ANA (B), anti‐dsDNA (C), anti‐fibronectin (D), anti‐vimentin (E), anti‐ AT1R (F), total IgG (G), and DSA (H) IgG levels in sera from allografted mice after 3 weeks of intravenous injections with vehicle (n = 13), ApoExo (n = 16), or apoptotic bodies (n = 17)

Figure 3

Apoptotic exosome‐like vesicles (ApoExo) triggers the recruitment of IL‐17⁺ RORγ⁺ γδT cells to vascular allografts. CD3⁺ T cell infiltrates in aortic allografts from allografted mice that underwent 3 weeks of injections with the vehicle (n = 7) or ApoExo (n = 8) were analyzed by flow cytometry for TCRβ, IL‐17, and RORγ expression. (A) Number of allograft infiltrating CD3⁺TCRβ⁺ cells for each condition. (B) Histogram showing detection of TCRβ⁺ cells. (C) Percentage of TCRβ⁺ cells in CD3⁺ allograft infiltrate for each condition. (D) Number of allograft infiltrating CD3⁺TCRγ⁺ cells for each condition. (E) Histogram showing detection of TCRγ⁺ cells. (F) Percentage of TCRγ⁺ cells in CD3⁺ allograft infiltrate for each condition. (G) Density plot for the detection of CD3⁺TCRγ⁺ cells that also express RORγ and/or IL‐17. (H) Percentage of RORγ⁺ cells in CD3⁺TCRγ⁺ allograft infiltrate for each condition. (I) Percentage of Il‐17⁺RORγ⁺ cells in CD3⁺TCRγ⁺ allograft infiltrate for each condition

Figure 4

Absence of γδT cells reduces allograft CD3⁺ T cell infiltration and IL‐17 expression, abrogates tertiary lymphoid structure (TLS) formation and reduces the formation of anti‐perlecan/LG3 and ANA. γδKO (n = 4) or wild‐type (n = 4) mice were injected with apoptotic exosome‐like vesicles (ApoExo) for 3 weeks posttransplantation: (A) Aortic allograft sections stained with H&E, CD20, CD3, AID, and IL‐17 (magnification: 5×; magnification of right inset panels: 20×). (B) Ratio intima/media in the allografts. (C) Mean number of TLS per allograft. Neointima‐media and perivascular quantification of CD20⁺ B cells (D), CD3⁺ T cells (E), AID (f), and IL‐17 (g) staining in each high‐power field of the allografts. Anti‐LG3 (h) and ANA (i) IgG levels in sera from γδKO or wild‐type allografted mice after 3 weeks of intravenous injections with ApoExo. Data were pooled from two independent experiments and expressed as means ± SEM. Comparison with the vehicle was done with a Student's t test

Figure 5

The proteasome activity within apoptotic exosome‐like vesicles (ApoExo) is required for induction of tertiary lymphoid structure (TLS) formation and for anti‐perlecan/LG3 production. Allografted mice were injected with ApoExo that were generated from serum‐starved murine epithelial cells (mECs) treated with bortezomib (n = 11) or the vehicle (n = 10) for 3 weeks posttransplantation: (A) Aortic allograft sections stained with H&E, CD20, CD3, AID, and IL‐17 (magnification: 5×, magnification of right inset panels: 20×). (B) Ratio intima/media ratio in the allografts. (C) Mean number of TLS per allograft. Neointima‐media and perivascular quantification of CD20⁺ B cells (D), CD3⁺ T cells (E), AID (F), and IL‐17 (G) staining in each high‐power field of the murine allografts. Anti‐LG3 (H) and ANA (I) IgG levels in sera from wild‐type allografted mice after 3 weeks of intravenous injections with ApoExo generated from serum‐starved mECs treated with bortezomib or the vehicle. Data were pooled from three independent experiments and expressed as means ± SEM. Comparison with the vehicle was done using a Student's t test

Figure 6

The proteasome activity within apoptotic exosome‐like vesicles (ApoExo) is required for the recruitment of RORγ⁺IL‐17⁺γδT cells to the allograft. Allografted mice were injected with ApoExo that were generated from serum‐starved murine epothelial cels treated with bortezomib (n = 10) or the vehicle (n = 9) for 3 weeks posttransplantation: CD3⁺TCRγ⁺ T cell aortic allograft infiltrates were analyzed by flow cytometry for IL‐17 and RORγ expression. (A) Number of allograft infiltrating of CD3⁺ expressing TCRβ or γ for each condition. (B) Density plot of CD3TCRβ⁺ and CD3⁺TCRγ⁺ cells expression of RORγ and IL‐17. (C) Percentage of CD3⁺TCRγ⁺ infiltrate expressing RORγ and or IL‐17 for each condition. Data were pooled from three independent experiments and expressed as means ± SEM