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We previously discovered suppressor T cell-derived, antigen (Ag)-specific exosomes inhibiting mouse hapten-induced contact sensitivity effector T cells by targeting antigen-presenting cells (APCs). These suppressive exosomes acted Ag-specifically due to a coating of antibody free light chains (FLC) from Ag-activated B1a cells. Current studies are aimed at determining if similar immune tolerance could be induced in cutaneous delayed-type hypersensitivity (DTH) to the protein Ag (ovalbumin, OVA). Intravenous administration of a high dose of OVA-coupled, syngeneic erythrocytes similarly induced CD3 + CD8 + suppressor T cells producing suppressive, miRNA-150-carrying exosomes, also coated with B1a cell-derived, OVA-specific FLC. Simultaneously, OVA-immunized B1a cells produced an exosome subpopulation, originally coated with Ag-specific FLC, that could be rendered suppressive by in vitro association with miRNA-150. Importantly, miRNA-150-carrying exosomes from both suppressor T cells and B1a cells efficiently induced prolonged DTH suppression after single systemic administration into actively immunized mice, with the strongest effect observed after oral treatment. Current studies also showed that OVA-specific FLC on suppressive exosomes bind OVA peptides suggesting that exosome-coating FLC target APCs by binding to peptide-Ag-major histocompatibility complexes. This renders APCs capable of inhibiting DTH effector T cells. Thus, our studies describe a novel immune tolerance mechanism mediated by FLC-coated, Ag-specific, miRNA-150-carrying exosomes that act on the APC and are particularly effective after oral administration.

Background: At present, the role of autologous cells as antigen carriers inducing immune tolerance is appreciated. Accordingly, intravenous administration of haptenated syngeneic mouse red blood cells (sMRBC) leads to hapten-specific suppression of contact hypersensitivity (CHS) in mice, mediated by light chain-coated extracellular vesicles (EVs). Subsequent studies suggested that mice intravenously administered with sMRBC alone may also generate regulatory EVs, revealing the possible self-tolerogenic potential of autologous erythrocytes., Objectives: The current study investigated the immune effects induced by mere intravenous administration of a high dose of sMRBC in mice., Methods: The self-tolerogenic potential of EVs was determined in a newly developed mouse model of delayed-type hypersensitivity (DTH) to sMRBC. The effects of EV's action on DTH effector cells were evaluated cytometrically. The suppressive activity of EVs, after coating with anti-hapten antibody light chains, was assessed in hapten-induced CHS in wild-type or miRNA-150 -/- mice., Results: Intravenous administration of sMRBC led to the generation of CD9 + CD81+ EVs that suppressed sMRBC-induced DTH in a miRNA-150-dependent manner. Furthermore, the treatment of DTH effector cells with sMRBC-induced EVs decreased the activation of T cells but enhanced their apoptosis. Finally, EVs coated with antibody light chains inhibited hapten-induced CHS., Conclusions and Clinical Relevance: The current study describes a newly discovered mechanism of self-tolerance induced by the intravenous delivery of a high dose of sMRBC that is mediated by EVs in a miRNA-150-dependent manner. This mechanism implies the concept of naturally occurring immune tolerance, presumably activated by overloading of the organism with altered self-antigens., (© 2019 John Wiley & Sons Ltd.)

We propose that there is a special B-1a B cell subset ("sB-1a" cells) that mediates linked processes very early after immunization to initiate cutaneous contact sensitivity (CS), delayed-type hypersensitivity (DTH), and immune resistance to pneumococcal pneumonia. Our published data indicate that in CS and DTH, these initiating processes are required for elicitation of the delayed onset and late-occurring classical T cell-mediated responses. sB-1a cells resemble memory B2 cells, as they are stimulated within 1 h of immunization and depend on T helper cytokines-uniquely IL-4 from hepatic iNKT cells--for activation and rapid migration from the peritoneal cavity to the spleen to secrete IgM antibody (Ab) and Ab-derived free light chains (FLCs) by only 1 day after immunization. Unlike conventional B-1a (cB-1a) cell-produced IgM natural Ab, IgM Ab produced by sB-1a cells has high Ag affinity owing to immunoglobulin V-region mutations induced by activation-induced cytidine deaminase (AID). The dominant cB-1a cells are increased in immunized AID-deficient mice but do not mediate initiation, CS, or pneumonia resistance because natural Ab has relatively low Ag affinity because of unmutated germ-line V regions. In CS and DTH, sB-1a IgM Ag affinity is sufficiently high to mediate complement activation for generation of C5a that, together with vasoactive mediators such as TNF-α released by FLC-sensitized mast cells, activate local endothelium for extravascular recruitment of effector T cells. We conclude by discussing the possibility of functional sB-1 cells in humans., (© 2015 New York Academy of Sciences.)

Background: Contact hypersensitivity (CHS) reaction induced by a topical application of hapten is a cell-mediated antigen-specific type of skin inflammation mediated by interaction of several subtypes of T cell subpopulations. Recently, it has been shown that antidepressant drugs inhibit CHS reaction, although the mechanism of this effect remains unknown. The aim of the present study was to investigate the effect of 2-week desipramine or fluoxetine administration on the CHS reaction induced by picryl chloride (PCL) application in B10.PL mice and in knock-out mice established on B10.PL background: TCRδ(-/-) mice lacking TCRγδ T lymphocytes; β2m(-/-) mice lacking CD8(+) T lymphocytes and CD1d(-/-) mice lacking CD1d dependent natural killer T (NKT) lymphocytes., Methods: B10.PL, TCRδ(-/-), β2m(-/-) and CD1d(-/-) mice were divided into six groups: 1) vehicle-treated negative control group; 2) desipramine-treated negative control group; 3) fluoxetine-treated negative control group; 4) vehicle and PCL-treated group (positive control group); 5) desipramine and PCL-treated group; and 6) fluoxetine and PCL-treated group. CHS to PCL was tested by evaluation of ear swelling. Metabolic activity of spleen and lymph node cells were estimated by MTT test., Results: The antidepressants significantly suppressed the CHS reaction in B10.PL mice: desipramine by 55% and fluoxetine by 42% compared to the positive control. This effect was even stronger in TCRδ(-/-) mice, in which fluoxetine reduced the ear swelling by 73% in comparison with the vehicle-treated positive control group. On the other hand, desipramine and fluoxetine did not inhibit CHS reaction in β2m(-/-) and CD1d(-/-) mice. Moreover, PCL increased metabolic and/or proliferative activity of splenocytes in all four strains of mice whereas the antidepressants decreased this activity of splenocytes in B10.PL, TCRδ(-/-) and CD1d(-/-) mice., Conclusion: The results of the present study show that lack of CD8(+) T cells or NKT cells abolishes the immunosuppressive effect of antidepressant drugs on PCL-induced CHS reaction in mice. These results suggest that antidepressant drug-induced inhibition of CHS reaction is connected with their inhibitory effect on ability of CD8(+) T cells and NKT cells to induce and/or escalate CHS reaction. TCRγδ cells seem not to be involved in antidepressant-induced suppression of CHS., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Soon after the discovery of T suppressor cells by Gershon in 1970, it was demonstrated that one subpopulation of these lymphocytes induced by i.v. hapten injection suppresses contact sensitivity response mediated by effector CD4+ or CD8+ T cells in mice through the release of soluble T suppressor factor (TsF) that acts antigen specifically. Our experiments showed that biologically active TsF is a complex entity consisting of two subfactors, one antigen specific and other non-specific, produced by differently induced populations of cells. In following years, we found that the antigen-specific subfactor is a light chain of IgM antibody that is produced by B1a lymphocytes. However, the exact nature of non-specific part remained a mystery for about 30 years. Our current studies characterized TsF as regulatory miRNA-150 carried by T suppressor cell-derived exosomes that are antigen specific due to a surface coat of IgM antibody light chains produced by B1a cells. The present communication briefly summarizes our studies on TsF that led to discovery of regulating miRNA that acts antigen specifically to suppress immune response.

Murine contact sensitivity (CS) reaction could be antigen-specifically regulated by T CD8(+) suppressor (Ts) lymphocytes releasing microRNA-150 in antibody light-chain-coated exosomes that were formerly suggested to suppress CS through action on macrophages (Mφ). The present studies investigated the role of Mφ in Ts cell-exosome-mediated antigen-specific suppression as well as modulation of Mφ antigen-presenting function in humoral and cellular immunity by suppressive exosomes. Mice depleted of Mφ by clodronate liposomes could not be tolerized and did not produce suppressive exosomes. Moreover, isolated T effector lymphocytes transferring CS were suppressed by exosomes only in the presence of Mφ, demonstrating the substantial role of Mφ in the generation and action of Ts cell regulatory exosomes. Further, significant decrease of number of splenic B cells producing trinitrophenyl (TNP) -specific antibodies with the alteration of the ratio of serum titres of IgM to IgG was observed in recipients of exosome-treated, antigen-pulsed Mφ and the significant suppression of CS was demonstrated in recipients of exosome-treated, TNP-conjugated Mφ. Additionally, exosome-pulsed, TNP-conjugated Mφ mediated suppression of CS in mice pre-treated with a low-dose of cyclophosphamide, suggesting de novo induction of T regulatory (Treg) lymphocytes. Treg cell involvement in the effector phase of the studied suppression mechanism was proved by unsuccessful tolerization of DEREG mice depleted of Treg lymphocytes. Furthermore, the inhibition of proliferation of CS effector cells cultured with exosome-treated Mφ in a transmembrane manner was observed. Our results demonstrated the essential role of Mφ in antigen-specific immune suppression mediated by Ts cell-derived exosomes and realized by induction of Treg lymphocytes and inhibition of T effector cell proliferation., (© 2015 John Wiley & Sons Ltd.)

Lymph node and spleen cells of mice doubly immunized by epicutaneous and intravenous hapten application produce a suppressive component that inhibits the action of the effector T cells that mediate contact sensitivity reactions. We recently re-investigated this phenomenon in an immunological system. CD8+ T lymphocyte-derived exosomes transferred suppressive miR-150 to the effector T cells antigen-specifically due to exosome surface coat of antibody light chains made by B1a lymphocytes. Extracellular RNA (exRNA) is protected from plasma RNases by carriage in exosomes or by chaperones. Exosome transfer of functional RNA to target cells is well described, whereas the mechanism of transfer of exRNA free of exosomes remains unclear. In the current study we describe extracellular miR-150, extracted from exosomes, yet still able to mediate antigen-specific suppression. We have determined that this was due to miR-150 association with antibody-coated exosomes produced by B1a cell companions of the effector T cells, which resulted in antigen-specific suppression of their function. Thus functional cell targeting by free exRNA can proceed by transfecting companion cell exosomes that then transfer RNA cargo to the acceptor cells. This contrasts with the classical view on release of RNA-containing exosomes from the multivesicular bodies for subsequent intercellular targeting. This new alternate pathway for transfer of exRNA between cells has distinct biological and immunological significance, and since most human blood exRNA is not in exosomes may be relevant to evaluation and treatment of diseases.

Cells of multicellular organisms exchange informative signals by diverse mechanisms. Recent findings uncovered the special role of extracellular vesicles, especially exosomes, in intercellular communication. Exosomes, present in all tested human bodily fluids, carry various functional compounds including proteins, lipids, and diverse RNA molecules. The composition of exosome cargo in vivo is likely formed by a regulated selection of specific components and can express the current status of the exosome-secreting cell. Therefore, particular emphasis is now placed on the extremely high potential of exosomes as essentially noninvasive prognostic and diagnostic biomarkers, but also as therapeutic nanocarriers, especially after the discovery that their cargo as well as cell-targeting specificity could be shaped in vitro. In addition, targeting the exosomes mediating pathological intercellular communication may also express high therapeutic potential. Hence, numerous studies are conducted to explore the profile and function of exosomes and their cargo in health and disease and to shape their properties to facilitate their clinical application. The present review summarizes the current knowledge on the role of exosomes in different physiological and pathological mechanisms of intercellular communication with a particular focus on the use of exosomes in the diagnosis and treatment of various inflammatory, cardiovascular, metabolic, and neurodegenerative disorders as well as malignant neoplasms.