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

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 (100 000 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 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.