Dysfunctional regulation of the immune response is at the root of inflammatory disorders such as autoimmune and allergic diseases. These disorders arise when the immune system fails to properly distinguish between self and non-self, or overreacts to normally harmless substances. A distinct increase of autoimmune disease prevalence has been identified, particularly in industrial countries, which cannot be explained by genetic changes but, most likely, by changes in the environment (Zhao et al., 2019, Miller, 2023). As many autoimmune diseases are generally diagnosed early in life, they can develop as lifelong chronic conditions, posing new financial and societal strains, and irrevocably changing quality of life (Logel et al., 2023). Therapies available to date only alleviate disease symptoms and can be accompanied by numerous side effects (Fugger et al., 2020). The treatment that will restore immune tolerance and provide a long-term drug-free remission is still elusive.

Among cellular therapy-based treatments, regulatory T cells (Tregs) have been demonstrated to be one of the most promising solutions. Treg cells induce immune tolerance through multiple mechanisms such as releasing anti-inflammatory cytokines IL-10, tumor growth factor − β (TGF- β), consuming IL-2 and consequently blocking T cell proliferation or by cell-to-cell contact through inhibitory receptors,such as the cytotoxic T lymphocyte antigen 4 (CTLA-4) (Onishi et al., 2008, Sakaguchi et al., 2009, Raffin et al., 2020). Their deficiency has been linked with multi-organ inflammation and auto-immunity (Park et al., 2020) which include expansion of autoreactive T cells and increased production of proinflammatory cytokines (Borna et al., 2022). In our previous work we showed that generating regulatory T cells in vitro is possible by co-cultivation of naïve T cells and tolerogenic dendritic cells (DCs), generated under the influence of Trichinella spiralis muscle larvae excretory-secretory (ES L1) products (Ilic et al., 2018). Tolerogenic DCs represent a link between immunomodulatory ES L1 products, which ameliorate experimental autoimmune encephalomyelitis (EAE), in an animal model of multiple sclerosis when applied in vivo (Radovic et al., 2015a), and the expansion of regulatory T cells involved in reducing inflammation (Xue et al., 2022). Under the influence of ES L1 products, DCs acquire semi-matured phenotypes, marked by reduced expression of major histocompatibility complex class II (MHC II) and costimulatory molecules such as CD83 and CD86. This effect coincides with decreased production of the proinflammatory cytokine IL-12, alongside increased secretion of anti-inflammatory and regulatory cytokines such as IL-10 and TGF-β (Ilic et al., 2018), all of which are key factors in fostering immune tolerance (Khan et al., 2022). ES L1 products consist of different molecules, some of which have been previously investigated (Cvetkovic et al., 2014, Kobpornchai et al., 2020), with the aim of identifying those with immunomodulatory capacity. To our knowledge, our group was the first to confirm that T. spiralis muscle larvae ES L1 products, among other components, contain extracellular vesicles (EVs) with immunomodulatory potential (Kosanović et al., 2019). However, it remained unclear whether TsEVs can induce a similar tolerogenic response in human DCs, as complete ES L1 products.

EVs are nano-sized structures surrounded by a lipid bilayer, released by all cell types during physiological and pathological conditions. As crucial mediators of intracellular communication, EVs transfer a complex set of biomolecules including proteins, nucleic acids, lipids and metabolites between cells within the same organism as well between different organisms (Mir and Goettsch, 2020, Cuesta et al., 2021). Determining this delivery by EVs has revealed their potential as a diagnostic tool and provided valuable insights into their capacity to modulate cellular responses, positioning them as promising candidates for therapeutic applications (Gomzikova et al., 2019, Kodam and Ullah, 2021). Several studies have explored how parasitic EVs hold promise as a therapeutic approach for addressing autoimmune disorders and allergic diseases. For instance, EVs from Taenia pisiformis induce M2 polarization in RAW264.7 macrophages by stimulating the production of anti-inflammatory cytokines IL-4, IL-10, and IL-13 (L. Q. Wang et al., 2020). Additionally, EVs derived from Heligmosmoides polygyrus have shown the capacity to suppress eosinophilia and reduce IL-33 receptor expression in mice treated with allergenic fungus extracts from Alternaria (Buck et al., 2014). Lastly, EVs from T. spiralis (TsEVs) display a potent immunomodulatory capacity, demonstrated by reducing pro-inflammatory cytokine secretion and neutrophil infiltration in in vivo treatment of colitis in mice (Yang et al., 2020a) and by inducing M2b macrophage polarization, as well as by inhibiting fibroblast activation (Wu et al., 2022). In this study, we demonstrated that TsEVs promote the generation of tolerogenic DCs with the ability to steer allogeneic T cells towards an anti-inflammatory and regulatory T cell response, in order to assess the therapeutic potential of TsEVs.