*Purpose: Treg cells make a variety of immunosuppressive products—cytokines such as IL10, TGFβ, and IL35, and enzymes such as the CD39/CD73 pair that convert extracellular ATP into the inhibitory molecule adenosine. Of the 3 cytokines, IL10 is known to be produced in an active, H₂0-soluble form, while we have found in mice that both TGFβ and IL35 were produced in exosome-associated, latent form. Our purpose was to test the hypothesis that allopeptide-specific human Treg will produce exosomal IL35 in a self HLA-restricted manner in vitro.

*Methods: Human allopeptide-specific Tregs were expanded from PBMCs of stable kidney transplant recipients in vitro under repetitive stimulation with mismatched donor HLA-DR allopeptide in the presence of low dose IL-2 over a period of 30-35 d. Culture supernatants were collected after the final stimulation cycle on ~d. 28[“3x Stim.”] and at the end of expansion and resting ~ d. 30[“Media”]and spun sequentially at 2000xg, 10,000xg for 10 minutes each, and finally 100,000 xg for 2 hr. Ultracentrifuge-enriched exosome fractions, and exosome-negative supernatants were assayed for the presence of IL35 components.

*Results: In addition to “latent” CD81-associated IL35 found in exosomes produced by allospecific Treg cells in mice, a similar CD81-associated IL35 response was found in indirect pathway T/NK cell cultures of allopeptide-stimulated PBMC. As shown in the Figure below a similar bias toward the p35 component was found in the human exosomes.

*Conclusions: This finding may be relevant to: a) tolerogenic immunotherapy in deceased donor organ transplantation, b) the known correlation of bi-directional regulation with favorable kidney transplant outcomes in living-related donor-recipient pairs, and c) ongoing clinical kidney transplant tolerance trials.

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