*Purpose: Interleukin-35 (IL35) is an immunosuppressive cytokine composed of Ebi3 and p35 subunits. Yet the form(s) that IL35 assumes and its role in infectious tolerance, remain elusive. Our aim was to discern the unique features of this unique cytokine, and its relevance to infectious transplantation tolerance.

*Methods: CBA-specific, IL35⁺ Tregs (Foxp3⁺) were induced in C57BL/6 mice, and all IL35 producers were identified on d.35 using flow and Image Stream cytometry, by nuclear expression of Ebi3TdTom gene reporter, plus Ebi3 and p35 proteins.

*Results: Curiously, both subunits of IL35 were displayed on the surface of tolerogen-specific Foxp3⁺ induced Treg cells, Foxp3^neg (iTr35) T cells and Breg (IL35⁺) cells. Furthermore, IL35 producers, although rare, secreted Ebi3 and p35 on extracellular vesicles (EV), targeting a 25-100 fold higher number of T and B lymphocytes, causing them to acquire surface IL35 in the absence of autochtonous production. Surface IL35 on these non-producers was associated with exhaustion markers PD1, LAG3, and TIM3, and acquisition was prevented when EV/exosome production was inhibited in producers, or if Ebi3 was genetically deleted in Tregs prior to tolerogenesis. Antibodies to either IL35 subunit (Ebi3 or p35) co-precipitated the tetraspannin CD81, a key EV component of lymphocytes. CD81 association was critically dependent upon di-sulfide bonds, as indicated by loss of IL35 binding when 2ME was used to abolish CD81 2⁰ structure.

*Conclusions: The unique ability of exosomal IL35 to induce IL35 production in conventional T and B cells, and to coat bystander lymphocytes, promoting exhaustion in, and secondary suppression by, non-Treg cells, identifies a novel mechanism of infectious tolerance.

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