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

ABSTRACT

Here we test the hypothesis that, like CD81-associated "latent" IL35, the transforming growth factor (TGF)ßlatency-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 TGFß 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 TGFßLAP associated with tetraspanins CD81,CD63, and CD9 by enzyme-linked immunosorbent assay; (b) GARP, critical to membrane association of TGFßLAP and to activation from its latent form, as well as various TGFß receptors; and (c) TGFß-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/TGFßLAP-coated extracellular vesicles. Like IL35 subunits, but unlike IL10, which was absent from ultracentrifuge pellets, GARP/TGFßLAP was mainly associated with CD81+ exosomes. sEV-bound GARP/TGFßLAP 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/TGFßLAP 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 TGFßLAP that, like exosomal IL35, can target "bystander" lymphocytes. This new finding implicates exosomal TGFßLAP along with Treg-derived GARP as part of the infectious tolerance network.