Extracellular Vesicle-associated GARP/TGFβ:LAP Mediates "Infectious" Allo-tolerance

William J Burlingham; Ewa Jankowska-Gan; John H Fechner; Christopher J Little; Jianxin Wang; Seungpyo Hong; Miraf Molla; Jeremy A Sullivan; David P Foley

doi:10.1097/TXD.0000000000001475

Extracellular Vesicle-associated GARP/TGFβ:LAP Mediates "Infectious" Allo-tolerance

Transplant Direct. 2023 May 24;9(6):e1475. doi: 10.1097/TXD.0000000000001475. eCollection 2023 Jun.

Authors

William J Burlingham¹, Ewa Jankowska-Gan¹, John H Fechner¹, Christopher J Little¹, Jianxin Wang², Seungpyo Hong^{2

3}, Miraf Molla¹, Jeremy A Sullivan⁴, David P Foley¹

Affiliations

¹ Division of Transplantation, Department of Surgery, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI.
² Wisconsin Center for NanoBioSystems, University of Wisconsin-Madison, Madison, WI.
³ Pharmaceutical Sciences Division, School of Pharmacy, University of Wisconsin-Madison, Madison, WI.
⁴ Department of Anesthesiology, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI.

Abstract

Here we test the hypothesis that, like CD81-associated "latent" IL35, the transforming growth factor (TGF)β:latency-associated peptide (LAP)/glycoprotein A repetitions predominant (GARP) complex was also tethered to small extracellular vesicles (sEVs), aka exosomes, produced by lymphocytes from allo-tolerized mice. Once these sEVs are taken up by conventional T cells, we also test whether TGFβ could be activated suppressing the local immune response.

Methods: C57BL/6 mice were tolerized by i.p. injection of CBA/J splenocytes followed by anti-CD40L/CD154 antibody treatment on days 0, 2, and 4. On day 35, spleen and lymph nodes were extracted and isolated lymphocytes were restimulated with sonicates of CBA splenocytes overnight. sEVs were extracted from culture supernatants by ultracentrifugation (100 000g) and assayed for (a) the presence of TGFβ:LAP associated with tetraspanins CD81,CD63, and CD9 by enzyme-linked immunosorbent assay; (b) GARP, critical to membrane association of TGFβ:LAP and to activation from its latent form, as well as various TGFβ receptors; and (c) TGFβ-dependent function in 1° and 2° immunosuppression of tetanus toxoid-immunized B6 splenocytes using trans-vivo delayed-type hypersensitivity assay.

Results: After tolerization, CBA-restimulated lymphocytes secreted GARP/TGFβ:LAP-coated extracellular vesicles. Like IL35 subunits, but unlike IL10, which was absent from ultracentrifuge pellets, GARP/TGFβ:LAP was mainly associated with CD81⁺ exosomes. sEV-bound GARP/TGFβ:LAP became active in both 1° and 2° immunosuppression, the latter requiring sEV uptake by "bystander" T cells and reexpression on the cell surface.

Conclusions: Like other immune-suppressive components of the Treg exosome, which are produced in a latent form, exosomal GARP/TGFβ:LAP produced by allo-specific regulatory T cells undergoes either immediate activation (1° suppression) or internalization by naive T cells, followed by surface reexpression and subsequent activation (2°), to become suppressive. Our results imply a membrane-associated form of TGFβ:LAP that, like exosomal IL35, can target "bystander" lymphocytes. This new finding implicates exosomal TGFβ:LAP along with Treg-derived GARP as part of the infectious tolerance network.