Orally Administered Exosomes Suppress Mouse Delayed-Type Hypersensitivity by Delivering miRNA-150 to Antigen-Primed Macrophage APC Targeted by Exosome-Surface Anti-Peptide Antibody Light Chains.

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.

Syngeneic red blood cell-induced extracellular vesicles suppress delayed-type hypersensitivity to self-antigens in mice.

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.

Inhibitory effect of antidepressant drugs on contact hypersensitivity reaction is connected with their suppressive effect on NKT and CD8(+) T cells but not on TCR delta T cells.

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; ß2m(-/-) mice lacking CD8(+) T lymphocytes and CD1d(-/-) mice lacking CD1d dependent natural killer T (NKT) lymphocytes. METHODS: B10.PL, TCRδ(-/-), ß2m(-/-) 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 ß2m(-/-) 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.

Exosomes as mediators of intercellular communication: clinical implications.

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.

Free Extracellular miRNA Functionally Targets Cells by Transfecting Exosomes from Their Companion Cells.

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.

Macrophages play an essential role in antigen-specific immune suppression mediated by T CD8⁺ cell-derived exosomes.

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.

From Mysterious Supernatant Entity to miRNA-150 in Antigen-Specific Exosomes: a History of Hapten-Specific T Suppressor Factor.

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.

Enhanced generation of reactive oxygen intermediates by suppressor T cell-derived exosome-treated macrophages.

Macrophages (Mφ) as efficient phagocytes able to present the antigen and playing an effector role induce and orchestrate the immune response also through the release of soluble factors. Recently described T CD8+ cell-derived suppressive exosomes carrying miRNA-150, that act antigen-specifically, seem to inhibit murine contact sensitivity reaction indirectly by affecting antigen presenting cells, especially Mφ. Present studies investigated the influence of suppressive exosomes on secretory activity of Mφ assessed as their ability to generate reactive oxygen intermediates (ROIs), nitric oxide, cytokines as well as their viability and expression of antigen phagocytosis and presentation markers. Interestingly, in vivo and in vitro treatment of Mφ with assayed hapten-specific exosomes affected only ROIs generation, significantly enhancing their production. Current results suggest that ROIs may participate in antigen-specific tolerance mechanism mediated by suppressive T lymphocyte-derived exosome-influenced Mφ, by inhibition of effector T cell proliferation and induction of T regulatory lymphocytes.

Antigen-specific, antibody-coated, exosome-like nanovesicles deliver suppressor T-cell microRNA-150 to effector T cells to inhibit contact sensitivity.

BACKGROUND: T-cell tolerance of allergic cutaneous contact sensitivity (CS) induced in mice by high doses of reactive hapten is mediated by suppressor cells that release antigen-specific suppressive nanovesicles. OBJECTIVE: We sought to determine the mechanism or mechanisms of immune suppression mediated by the nanovesicles. METHODS: T-cell tolerance was induced by means of intravenous injection of hapten conjugated to self-antigens of syngeneic erythrocytes and subsequent contact immunization with the same hapten. Lymph node and spleen cells from tolerized or control donors were harvested and cultured to produce a supernatant containing suppressive nanovesicles that were isolated from the tolerized mice for testing in active and adoptive cell-transfer models of CS. RESULTS: Tolerance was shown due to exosome-like nanovesicles in the supernatants of CD8(+) suppressor T cells that were not regulatory T cells. Antigen specificity of the suppressive nanovesicles was conferred by a surface coat of antibody light chains or possibly whole antibody, allowing targeted delivery of selected inhibitory microRNA (miRNA)-150 to CS effector T cells. Nanovesicles also inhibited CS in actively sensitized mice after systemic injection at the peak of the responses. The role of antibody and miRNA-150 was established by tolerizing either panimmunoglobulin-deficient JH(-/-) or miRNA-150(-/-) mice that produced nonsuppressive nanovesicles. These nanovesicles could be made suppressive by adding antigen-specific antibody light chains or miRNA-150, respectively. CONCLUSIONS: This is the first example of T-cell regulation through systemic transit of exosome-like nanovesicles delivering a chosen inhibitory miRNA to target effector T cells in an antigen-specific manner by a surface coating of antibody light chains.

Down-regulation of macrophage immune activity by natural CD8+ regulatory T cells.

To evaluate the influence of natural regulatory CD8+ T cells on macrophages we investigated in vitro production of cytokines, reactive oxygen intermediates (ROIs) and expression of CD80 surface costimulatory molecules by macrophages (MF) of wild type (WT) B10PL and syngeneic knock-out (KO) strains, TCRalpha-/-, beta2m-/- and CD1d-/- mice. MF of TCR alpha-/- (CD4- and CD8-) and beta2m-/- (CD8-) animals produced higher levels ofTNF-a, IL-6, IL-12 and ROIs and showed increased expression of CD80 costimulatory molecules in comparison to MF of WT or CD1d-/- (NKT-) mice. When MF of these strains were conjugated with TNP hapten and injected i.v. into WT mice to test either induction of contact sensitivity (CS) or tolerance, only TNP-MF of TCRalpha-/- and beta2m-/- animals induced a significant CS reaction, while cells of WT and CD1d-/- strains were tolerogenic. MF of the tested strains can be classified functionally as resembling either proinflammatory (TCRalpha-/- and beta2m-/-mice) or immunosuppressive (WT and CD1d-/-) phenotypes. We suggest the presence of an in vivo regulatory loop in which innate CD8+ Treg cells control the transition between MF phenotypes and thus adjust the magnitude of the inflammatory response to strictly local requirements.

Inhibition of 2,4-dinitrofluorobenzene-induced contact hypersensitivity reaction by antidepressant drugs.

BACKGROUND: Contact hypersensitivity (CHS) induced by a topical application of hapten - 2,4-dinitrofluorobenzene (DNFB), is a T cytotoxic (Tc)1-cell-mediated antigen-specific type of skin inflammation. Recently, it has been shown that antidepressant drugs inhibit the T helper (Th)1-mediated CHS reaction induced by picryl chloride. The aim of present study was to establish the effect of two-week desipramine or fluoxetine administration on the CHS reaction induced by DNFB. METHODS: Balb/c (H-2(d)) male 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-treated DNFB group (positive control group); 5) desipramine-treated DNFB group; 6) fluoxetine-treated DNFB group. T lymphocytes proliferation was determined by incorporation of [(3)H]-thymidine to DNA of concanavalin A stimulated cells. ELISA test was used for estimation of cytokines production. RESULTS: The antidepressants significantly suppressed the CHS reaction mediated by Tc1 cells: desipramine by 55% and fluoxetine by 54% compared to the positive control. Moreover, the antidepressants decreased the proliferative activity of splenocytes and the ability of splenocytes to produce interleukin (IL)-6 and interferon (IFN)-γ and increased IL-10 production by the lymph node (LN) cells of DNFB-treated mice. CONCLUSION: The results of the present study show that the Tc1-dependent reactivity to DNFB is significantly suppressed by antidepressant drugs, which suggests their inhibitory effect on Tc1 mediated immunity.

Inhibitory effect of antidepressant drugs on contact hypersensitivity reaction.

BACKGROUND: Contact hypersensitivity (CS) reaction in the skin is T-cell mediated immune reaction which plays a major role in the pathogenesis and chronicity of various inflammatory skin disorders and, like other delayed-type hypersensitivity (DTH) reactions, affords immunity against tumor cells and microbes. CS response is a self-limiting reaction, and interleukin (IL)-10 is considered to be a natural suppressant of cutaneous inflammatory response. Recently, it has been demonstrated that major depression is related to activation of the inflammatory response and elevation of some parameters of cell-mediated immunity. It has been suggested that such activation of the immune system may play a role in etiology of depression. If this immunoactivation is involved in etiology of depression, one would expect that antidepressant agents may have negative immunoregulatory effects. To the best of our knowledge, the effect of antidepressants on contact hypersensitivity has not been studied. METHODS: The aim of the present study was to establish the effect of prolonged desipramine or fluoxetine treatment on CS reaction to picryl chloride. RESULTS: Antidepressants significantly suppressed CS reaction, fluoxetine by 53% whereas desipramine by 47% compared to positive control. Moreover, desipramine and fluoxetine decreased relative weight of auxillary lymph nodes. Desipramine decreased also relative weight of inguinal lymph nodes and spleens whereas desipramine and fluoxetine increased production of IL-10 in comparison to positive control. CONCLUSION: The observed effect of antidepressant drugs on CS reaction is consistent with the hypothesis that T-cell mediated immunity is targeted by antidepressants.

Epicutaneous immunization with DNP-BSA induces CD4+ CD25+ Treg cells that inhibit Tc1-mediated CS.

As we have shown previously that protein antigen applied epicutaneously (EC) in mice inhibits TNP-specific Th1-mediated contact sensitivity (CS), we postulated that the maneuver of EC immunization might also suppress Tc1-dependent CS response. Here we showed that EC immunization of normal mice with 2,4-dinitrophenylated bovine serum albumin (DNP-BSA) applied on the skin in the form of a patch induces a state of subsequent unresponsiveness due to regulatory T cells (Treg) that inhibited sensitization and elicitation of effector T-cell responses. Suppression is transferable in vivo by TCRαß(+) CD4(+) CD25(+) lymphocytes harvested from lymph nodes (LNs) of skin-patched animals. Flow cytometry revealed that EC immunization with DNP-BSA increased TCRαß(+) CD4(+) CD25(+) FoxP3(+) lymphocytes in subcutaneous LNs, suggesting that observed suppression was mediated by Treg cells. Further, in vitro experiments showed that EC immunization with DNP-BSA prior to 1-fluoro-2,4-dinitrobenzen sensitization suppressed LN cell proliferation and inhibited production of TNF-α, IL-12 and IFN-γ. Using a transwell system or anti-CTLA-4 mAb, we found that EC induced suppression required direct Treg-effector cell contact and is CTLA-4-dependent.

Epicutaneous immunization with protein antigen induces antigen-non-specific suppression of CD8 T cell mediated contact sensitivity.

BACKGROUND: Allergic contact dermatitis (ACD) resulting from exposure to low molecular weight chemicals is a common clinical condition in industrialized countries and can be mediated by either Th1 or Tc1 lymphocytes. The animal model of contact sensitivity (CS) is commonly used to study ACD in mice and helps to test new therapeutics. We have previously shown that epicutaneous (EC) immunization with TNP-Ig prior to hapten sensitization inhibits Th1-mediated CS and observed that the suppression is mediated by TCRαß(+) CD4(+) CD8(+) cells and is TGF-ß dependent. More recently we have shown that EC immunization with DNP-BSA induces TCRαß(+) CD4(+) CD25(+) FoxP3(+) T regulatory (Treg) cells that suppress Tc1-mediated CS. METHODS: Animal model of contact sensitivity was used to study skin-induced suppression. RESULTS: Current work employing Tc1-mediated CS shows that skin-induced suppression is dose-dependent and declines with time. Experiments with the four non-cross-reacting antigens 2,4-dinitrophenylated bovine serum albumin (DNP-BSA), ovalbumin (OVA), myelin basic protein (MBP) and immunoglobulins conjugated with oxazolone (OX-Ig) employing models of active suppression, "transfer in" and "transfer out" protocols showed that EC immunization with any tested protein antigen inhibits CS response suggesting lack of antigen-specificity of the investigated phenomenon. CONCLUSION: The ease of EC generation of antigen-non-specific regulatory cells may have important implications for designing therapeutic schemes aimed at modulating immune responses.

[Immunoregulation by interference RNA (iRNA) - mechanisms, role, perspective]. / Immunoregulacja poprzez interferencyjny RNA ­ mechanizmy, rola, perspektywy.

The functioning of an organism depends on the precise control mechanisms, constantly adjusted to the actual state. Therefore, there is a need for efficient communication between both adjacent and distant cells, which may be executed by proteins such as hormones, neurotransmitters and cytokines. Recently another means of regulation has emerged - short regulatory RNAs (srRNAs). Although discovered only a couple of years ago, the mechanism of RNA interference has already become a topic of thousands of publications, defining its roles in both physiological and pathological processes, such as cancerogenesis and autoimmunization. RNAs regulating cell function may be coded in its genome (both exons and introns) or be introduced from the external environment. In mammals microRNAs (miRNAs) cooperate with proteins from the Ago/PIWI family to form effector ribonucleoprotein complexes, and owing to their complementarity to the target mRNA, control genes' expression at the posttranscriptional level, either through the suppression of mRNA translation or through mRNA degradation. SrRNAs are crucial regulators throughout the development of immune cells, starting from hematopoietic stem cells, up to the effector cells of the adaptive immune response. Moreover, some of the regulatory cells perform their function by releasing miRNAs, which are then transported to the target cells, possibly enclosed in the exosomes.

Role of TLR ligands in epicutaneously induced contrasuppression.

Our previous work showed that epicutaneous (EC) immunization in mice with protein antigen (Ag) induced an Ag-independent unresponsiveness mediated by suppressor CD4(+)8(+) T cells (Ts), which inhibited contact hypersensitivity (CS). Simultaneous EC immunization with Ag and various Toll-like receptor (TLR) ligands reversed skin-induced suppression. Our present study shows that this process activates Ag-specific T contrasuppressor (Tcs) cells and leads to the protection of CS effector T cells from suppression. Epicutaneous immunization with Ag and the TLR4 ligand lipopolysaccharide (LPS) led to a significant increase in IFN-gamma production by lymph node and spleen cells. Ag and TLR ligands, like LPS, CpG or lipoteichoic acid did not need to be applied concomitantly to the skin. An identical contrasuppressive effect was observed when the Ag and TLR ligands were deposited on distant skin areas, suggesting that both the generation of Ts and Tcs are independent. To corroborate this finding, we used a model system that uses macrophages (Mf) as Ag-presenting cells. Mf labeled in vitro with Ag (Mf-Ag) induced, upon intravenous (iv) administration, an unresponsiveness reaction that was mediated by Ts cells. When treated simultaneously with LPS-treated Mf (Mf-Ag-LPS), a TLR-ligand could induce CS. Both the Ag and the LPS signal could be uncoupled i.e., Mf-Ag and Mf-LPS given at separate time points (with an 1 h interval between injections) induced immunity.We also found that LPS-treated Mf also produced significant amounts of IL-12, a cytokine that has well-known anti-tolerogenic properties. Our experiments suggest that reversal of EC-induced suppression by TLR-ligands may be a potential tool to increase the immunogenicity of weakly immunogenic antigens.

Influence of cyclophosphamide and its metabolic products on the activity of peritoneal macrophages in mice.

2,4,6-Trinitrophenyl (TNP) hapten-labeled peritoneal macrophages (Mf) given intravenously (iv) to recipients are poor inducers of contact sensitivity (CS) reactions unless Mf donors are pretreated with low doses of cyclophosphamide (CY). In vivo CY is converted into active alkylating metabolites, phosphoramide mustard (PM) and acrolein (ACR). Our experiments aimed to test how in vitro treatment of non-immunogenic Mf with different concentrations (10(-5) to 10(-7) M) of CY metabolites will influence their immunogenicity and other biological functions. Instead of chemically unstable PM, we used structurally and functionally similar nitrogen mustard (NM). Our experiments show that treatment of Mf with ACR or NM stimulates the in vitro production of pro-inflammatory IL-6 and IL-12 and down-regulates anti-inflammatory IL-10 and TGF-beta cytokines. In vivo non-immunogenic TNP-Mf become capable of inducing CS reactions in two situations: first, after treatment with NM or ACR and second, when cell recipients are received iv before Mf transfer of monoclonal antibodies against IL-10 and/or TGF-beta (500 mug per animal). Treatment with NM, but not with ACR, was also an efficient stimulus for production by Mf of significantly increased levels of reactive oxygen intermediates (ROIs). In summary, our experiments show that CY metabolites can significantly increase the specific immune response as well as nonspecific innate reaction (ROIs production) and support the notion that CY and its metabolites can be a promising accessory tool when upregulation of the immune response is desired.

Epicutaneous immunization with protein antigen in the presence of TLR4 ligand induces TCR alpha beta+CD4+ T contrasuppressor cells that reverse skin-induced suppression of Th1-mediated contact sensitivity.

Our previous work showed that epicutaneous (EC) immunization of mice with different protein Ags applied on the skin in the form of a patch induces a state of subsequent Ag-nonspecific unresponsiveness due to suppressor CD4+8+ T cells (Ts) that inhibit Th1-mediated contact sensitivity (CS) reactions via released TGF-beta. In the present work we show that EC immunization with Ag together with the TLR4 ligand LPS induced cells that could prevent suppression by the Ag-nonspecific Ts. These up-regulatory cells, called contrasuppressor T cells (Tcs), belong to a population of Ag-specific TCRalphabeta CD4+ lymphocytes and are different from Th1 CD4+ cells that mediate the CS reaction. Experiments using knockout mice showed that EC induced contrasuppression is MyD88, INF-gamma, and IL-12 dependent, whereas IL-6 is not involved in this phenomenon. Additional experiments with anti-IFN-gamma mAb showed that IFN-gamma is required for induction of Tcs cells but does not play a crucial role in the effector phase of contrasuppression. Additionally, treatment of CS effector cells with rIL-12 makes them resistant to EC induced suppression without affecting Ts cells, whereas IL-12 neutralization in vitro abrogates contrasuppression. These data show that IL-12 is indeed involved in the effector phase of EC induced contrasuppression and that this cytokine does not act directly on Ts cells. The mechanism of action of Tcs protects Th1 effector cells mediating CS from the nonspecific Ts, leaving suppression to other Ags intact. Ts and Tcs cells do not influence each other and can be induced simultaneously in the same animal.

Mesenteric lymph node Tgammadelta cells induced by gastrectomy in mice suppress cell-mediated immune response in vitro via released TGF-beta.

We have shown previously that gastrectomy, but not laparotomy alone, severely impairs contact sensitivity responses in vivo and selectively alters cell trafficking in gut associated lymphatic tissue. Here, we investigate the immunological role of different subpopulations of mesenteric lymph node cells (MLNCs) in the inhibition of contact sensitivity as well as their suppressive mechanisms. Suppressive cells were isolated from the mesenteric lymph nodes of gastrectomized mice and were added to cultures of lymphocytes from mice immunized with trinitrophenyl-chloride. These MLNCs inhibited the proliferation of sensitized lymphocytes in response to antigen. Depletion experiments revealed that the suppressive MLNCs are Tgammadelta+ cells, but not Talphabeta+ cells. Neutralizing antibodies to IL-4, IL-10, and tissue growth factor-beta (TGF-beta) revealed that suppression was dependent on TGF-beta, but not the other cytokines. We conclude that surgical stress induced by gastrectomy causes accumulation of Tgammadelta+ lymphocytes in gut associated lymphatic tissue and that these cells suppress the cell-mediated response in vitro in an antigen-non specific manner via TGF-beta. This cytokine can possibly prevent in vivo the development of autoimmune responses following severe tissue trauma in the gastrointestinal tract.