Skip to main content
NCBI home page
Immunology logoLink to Immunology
. 2015 Apr 27;146(1):23–32. doi: 10.1111/imm.12466

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

Katarzyna Nazimek 1, Wlodzimierz Ptak 1, Bernadeta Nowak 1, Maria Ptak 1, Philip W Askenase 2, Krzysztof Bryniarski 1,✉
PMCID: PMC4552498  PMID: 25808106

Abstract

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.

Keywords: exosomes, immune suppression, macrophages, T CD8+ suppressor lymphocytes, T regulatory lymphocytes

Introduction

Recently, we have reported the murine antigen-specific immune suppression mechanism mediated by T CD8+ suppressor (Ts) lymphocyte-derived exosomes carrying microRNA-150 (miRNA-150) and coated with B1 cell-released antigen-specific antibody light-chains.1–4 The exact characteristics of the studied exosomes were also described.4 Ts cells that do not express the FoxP3 marker are activated to release regulatory miRNA-150 in exosomes by intravenous administration of a high dose of hapten-labelled syngeneic erythrocytes. Murine tolerized donors are also contact immunized with the same hapten, which activates B1 cells to generate hapten-specific antibody light-chains that bind to the exosome surface, enabling them to bind antigen. This capacity of exosomes allows for their specific isolation and purification by antigen-affinity column chromatography.4 Interestingly, intravenous administration of a high dose of syngeneic erythrocytes (untreated with hapten) followed by skin painting with hapten-free vehicle results in the generation of antigen-non-specific and non-binding exosomes that we named Sham factor (SHAM-F).4 SHAM-F exosomes also contain miRNA-150 and are able to antigen-non-specifically suppress the HT-2 cell line responsiveness to IL-2 (K. Bryniarski, P.W. Askenase, unpublished results), analogously to hapten-specific exosomes and exosomes generated by Ts cells from tolerized immunoglobulin-deficient JH−/− knock-out (KO) mice.4 The enigmatic mechanism of SHAM-F exosome formation and action (originally possibly associated with regulation of haematopoiesis) remains our current research interest.

The regulatory activity of hapten-specific exosomes containing miRNA-150 has been studied so far in the murine hapten-induced contact sensitivity (CS) response. Ts cell-derived exosomes were shown to be able to inhibit the induction and elicitation phases of the CS reaction, to suppress the adoptive transfer of CS effector cells as well as to alleviate the clinical symptoms of active allergy.1,4 However, the exact mechanism of exosome regulatory action remains unclear and recent data suggest that exosomes act on CS effector T lymphocytes indirectly through antigen-presenting cells.

Macrophages (Mφ) are widely distributed in almost all body tissues and after activation can professionally present antigen as antigen-presenting cells to orchestrate the immune response. Mφ are involved as antigen-presenting cells and effector cells in delayed-type hypersensitivity reactions, including CS, as well as being able to induce a humoral immune response to corpuscular antigen. Previous studies reported the ability of Mφ to bind suppressive exosomes5 and suggested their important role in the currently investigated suppression mechanism.6–12

Current studies aimed to investigate the role of Mφ in Ts cell-derived exosome-mediated suppression of the immune response as well as to determine the ability of assayed exosomes to modulate the antigen-presenting function of Mφ in the induction of humoral and cellular immunity.

Materials and methods

Mice

CBA/J mice were from the breeding unit of the Department of Immunology, Jagiellonian University Medical College; BALB/c mice were from the National Cancer Institute, Jackson Laboratories (Bar Harbor, ME); and DEREG mice depleted of T FoxP3+ regulatory cells by diphtheria toxin intravenous injections (verified by flow cytofluorometry) were from Tim Sparwasser (Institute of Infection Immunology, Hannover, Germany).13 Ten-week-old mice were fed autoclaved food and water. All experiments were conducted according to the guidelines of both Jagiellonian and Yale Universities (No 39/2011 and 154/2013).

Haptens, antigens and proteins

Lyophilized guinea pig complement (Biomed, Lublin, Poland), sheep erythrocytes (SRBC) (Agricultural University, Krakow, Poland), trinitrophenyl (hapten) -conjugated mouse γ-globulins (TNP42-MGG) prepared by us as described previously,14 picryl chloride (PCL, TNP-Cl) (Chemtronix, Swannanoa, NC), and trinitrobenzene sulphonic acid (TNBSA) (Eastman Chemicals, Rochester, NY) were used.

Culture media, buffers and reagents

Mishell–Dutton medium, RPMI-1640, minimal essential medium with amino acids, HEPES, cacodylic buffer, Tris–HCl buffer, 2-mercaptoethanol, cyclophosphamide, DMSO, mineral oil heavy fraction (Sigma, St Louis, MO), fetal calf serum, Dulbecco’s phosphate-buffered saline (DPBS), thioglycollate medium, penicillin/streptomycin, sodium pyruvate, l-glutamine (Gibco Life Technologies, Grand Island, NY), acetone, ethanol, glucose (P.O.Ch., Gliwice, Poland), heparin (Polfa, Warszawa, Poland), EDTA (BDH, Poole, UK), extra virgin olive oil (Basso Fedelee Figli, San Michele di Serino, Italy), [3H]thymidine (Lacomed, Rez, Czech Republic), clodronate liposomes and control liposomes15,16 (Department of Molecular Cell Biology, Vrije University, Amsterdam, the Netherlands) were used.

Exosome generation and harvest

The Ts lymphocytes were activated as described previously4,17 by single (mice with depleted populations of cells) or double intravenous injections of 0·2 ml of a 10% DPBS suspension of TNP-labelled syngeneic erythrocytes (TNP-MRBC)4 on days 0 and 4, which was followed by contact immunization on shaved abdomen skin with 0·15 ml of 5% PCL solution in ethanol : acetone [3 : 1 volume/volume (v/v)] on day 9. On day 11, spleens and lymph nodes containing activated Ts cells were collected from tolerized mice and single cell suspensions were cultured in fetal calf serum-free Mishell–Dutton medium at a concentration of 2 × 107 cells/ml for 48 hr.4 The resulting supernatant was subsequently centrifuged at 300 g and 10 000 g for 10 min, filtered through 0·45-, 0·22- and 0·1-μm molecular filters and then ultracentrifuged twice at 100 000 g for 70 min at 4°.4 The resulting pellet was resuspended in DPBS4 and used as TNP-specific suppressive exosomes.

For SHAM-F exosomes,4 unlabelled MRBC treated as for hapten conjugation were injected into naive mice that were then skin painted with vehicle without hapten. This was followed by spleen and lymph node cell harvest and culture as above.

Negative factor control exosomes were obtained from ultracentrifuged supernatants of cultures from lymph node and spleen cells of naive mice, and processed as above.

Harvest of Mφ

Peritoneal Mφ were induced by intraperitoneal injection of either 1 ml of mineral oil or, for humoral immunity assays, 2 ml of thioglycollate medium.18 Five days later, Mφ were harvested by washing the peritoneal cavity with ice-cold DPBS containing heparin (5 U/ml) from naive or PCL-contact immunized mice. Splenic Mφ were separated from single-cell suspension of PCL-immunized donor spleens by their adherence to plastic Petri dishes (60 min at 37°) followed by their harvest by incubation on ice with ice-cold 0·02% EDTA in DPBS for 10 min. Then, peritoneal or splenic Mφ were treated in vitro with suppressive or negative factor (NF) control exosomes in a dose of 10 μl of exosome suspension in DPBS (about 4 × 109 pelleted exosomes, as estimated by Nanoparticle Tracking Analysis)4 per 1 × 106 cells for 30 min in a 37° water-bath followed by washing of excessive vesicles at 300 g. In some cases, exosome-pulsed Mφ were labelled with TNP derivative by incubating them for 10 min at room temperature in darkness with TNBSA in DPBS solution (2 mg/ml) at a ratio of 2 mg of TNBSA per 1 × 108 Mφ. For humoral assays, exosome-pulsed Mφ were fed with TNP-labelled SRBC by incubation for 30 min at 37° at a ratio of 10 TNP-SRBC per Mφ, followed by removal of non-phagocytosed erythrocytes through osmotic shock and intraperitoneal injection (4 × 106 Mφ per mouse) of Mφ into naive mice. A detailed methodology of plaque-forming and haemagglutination assays used for humoral immunity assessment was recently described.18 To measure IgG titre, sera were pre-incubated with 0·15 m 2-mercaptoethanol.18 The percentage of Mφ in isolated populations of cells evaluated in a non-specific esterase assay19 in each case exceeded 95%.

Active sensitization and adoptive transfer of CS effector cells

Naive mice were actively contact sensitized with 150 μl of 5% PCL in an ethanol : acetone mixture (3 : 1 v/v) on shaved abdomen skin. After 5 days, a CS response was elicited by application of 10 μl of 0·4% PCL in acetone : olive oil (1 : 1 v/v) on each side of both ears (challenge). Twenty-four hours later, the CS reaction was measured as ear swelling response with an engineer’s micrometer (Mitutoyo, Japan) by a blinded observer.4,20 It is noteworthy that measurement of ear swelling for CS reaction assessment correlates strongly with other methods and allows for repetition of evaluation.21

For adoptive transfer, CS effector cells were harvested from lymph nodes and spleens of contact-sensitized mice (see above), treated with suppressive or control exosomes for 30 min in a 37° water-bath and after washing at 300 g were transferred intravenously into naive recipients (7 × 107 cells per mouse) that were immediately challenged to elicit a CS reaction, which was measured 24 hr later.4,20,21 When necessary, T effector lymphocytes were isolated by triple separation on a nylon wool column10,22 and eventually supplemented with peritoneal or splenic Mφ from contact-sensitized donors. In some cases, lymph node and spleen CS effector cells (devoid of Mφ) were collected from mice that had been depleted of Mφ 24 hr earlier by intraperitoneal injection of 200 μl of clodronate liposomes.15,16

The TNP-Mφ were transferred intravenously (5 × 106 cells per recipient) into either naive recipients or mice treated intravenously 24 hr earlier with a low dose of cyclophosphamide (50 mg/kg of body mass). Seven days later the CS reaction was elicited and then measured as described above.

Depletion of Mφ or T regulatory lymphocytes

During the tolerogenesis procedure CBA/J mice were depleted of Mφ by single injection of 200 μl of clodronate liposomes15,16 delivered either intravenously 24 hr before TNP-MRBC administration or intraperitoneally 24 hr before contact sensitization.

DEREG mice13 were depleted of FoxP3+ regulatory T (Treg) cells by double intravenous diphtheria toxin injections (1 μg per mouse) 2 days and 1 day before the administration of TNP-MRBC. The efficiency of the depletion procedure was verified by flow cytofluorometric analysis after co-staining of lymph node, spleen and blood cells with fluoresceinated anti-CD4, anti-CD8 and anti-FoxP3 monoclonal antibodies.

Transmembrane cell culture

CS effector cells were cultured in triplicates in flat-bottom 24-well plates (1 × 106 per well) in the presence of antigen (25 μl of 1 mg/ml TNP-MGG solution). Mφ (1 × 105 cells per well or insert) were added directly to the wells or were placed into the inserts with semi-permeable membrane (Nunc, Roskilde, Denmark; cat. No 136730). Then the suppressive exosomes were added (1 × 109 exosomes per well) into wells or inserts. After 24 hr, inserts were removed and [3H]thymidine was added to each well (1 μCi per well).23 The culture was continued for the next 18 hr in standard conditions and the radioactivity of incorporated [3H]thymidine was measured in a scintillation counter.

Statistical analysis

Each experiment was carried out two or three times and all groups consisted of five mice. The results of representative experiments are shown in the figures. Average value of the non-specific increase of ear thickness due to chemical irritation by vehicle and hapten in challenged but not sensitized littermates was subtracted from average values in the experimental groups to obtain a net swelling value (Δ). In the case of in vitro tests, i.e. haemagglutination and proliferation assays, the results of repeated experiments were pooled for statistical analysis. Statistical significance of the data was estimated (after control for the meeting of test assumptions) in one-way analysis of variance with post hoc RIR Tukey test or, in the case of haemagglutination assay, a chi-squared test and confirmed in non-parametric equivalent tests.

Results

Treatment of Mφ with suppressive exosomes impairs their ability to induce humoral immune response

To assess the capacity of suppressive exosomes to influence the ability of Mφ to induce a humoral immune response to corpuscular antigen, Mφ pre-treated with TNP-specific or SHAM-F exosomes and then pulsed with TNP-labelled SRBC were administered intraperitoneally into naive recipients, from which blood sera and spleens were separately collected 7 days later. A significantly decreased number of antigen-specific antibody (plaque) -forming cells, evaluated in plaque-forming assay,18,23–25 was observed in spleens of recipients of TNP-specific and SHAM-F exosome-pulsed Mφ (Fig.1a, groups B and C), whereas pre-treatment of Mφ with NF exosomes had no effect (Fig.1a, group D). Further, estimated in a direct haemagglutination assay,18,24 an increased specific IgM to IgG antibody titre ratio was observed in sera of mice given suppressive exosome-pulsed Mφ (Fig.1b, groups B and C versus A and D). Both results confirmed the reduced ability of Mφ to induce humoral immunity after treatment with suppressive exosomes.

Figure 1.

Figure 1

Open in a new tab

Exosome treatment impairs macrophage ability to induce humoral immune response. Macrophages were incubated with trinitrophenyl (TNP) -specific, Sham factor (antigen-non-specific suppressor factor) (SHAM-F) or negative factor (NF) exosomes for 30 min, then with TNP-sheep red blood cells (SRBC) for an additional 30 min and, after removal of excessive exosomes and SRBC, were administered intraperitoneally into naive recipients. Seven days later, sera and spleens were individually collected from recipient mice to assess humoral immune response in haemagglutination (b) and plaque-forming (a) assays, respectively. (a) Decreased number of plaque-forming cells in spleens of recipients of exosome-pulsed macrophages fed with corpuscular antigen (groups B and C versus groups A and D). (b) Altered ratio of specific IgM to IgG antibody titres in sera of suppressive exosome-treated, antigen-fed macrophages (groups B and C versus A and D). **P < 0·01; ***P < 0·001.

Macrophages are required for exosome-mediated suppression of CS response

To determine the possible role of Mφ in exosome-mediated suppression, these cells were depleted in vivo by a single injection of clodronate liposomes15,16 into mice that were then tolerized before active CS elicitation. Depletion of Mφ before either TNP-MRBC administration or PCL contact immunization resulted in significant blockage of CS suppression (Fig.2a, groups C and E versus B), whereas administration of control ‘empty’ liposomes did not alter the suppression (Fig.2a, groups D and F). After CS estimation, lymph nodes and spleens were collected from all mice from each group and single cell suspensions were cultured identically as Ts cells for 48 hr. The resulting supernatants were then processed as for suppressive exosome isolation and ultracentrifuged pelleted material was tested for its potential biological activity. Recipients of CS effector cells incubated with this pelleted material developed not affected CS response (Fig.2b, groups C and E versus D and F). Hence, we concluded that Mφ are required for both the generation and suppressive action of assayed Ts cell-derived exosomes.

Figure 2.

Figure 2

Open in a new tab

Exosome-mediated suppression of contact sensitivity (CS) reaction is ineffective in the absence of macrophages. Mice received clodronate liposomes either intravenously 24 hr before trinitrophenyl-labelled mouse red blood cell (TNP-MRBC) injection or intraperitoneally 24 hr before picryl chloride (PCL) contact immunization. Five days later CS reaction was elicited by ear challenge and measured 24 hr later as ear swelling response (a). Then, lymph nodes and spleens were collected from mice of each group and cultured as for exosome generation. Ultracentrifuged pellets of resulting supernatants were tested in adoptive transfer of CS effector cells (b). Subsequently, macrophage role in exosome-mediated suppression was tested in adoptive transfer of exosome-treated CS effector cells depleted of macrophages, collected from donors pre-treated with clodronate liposomes (c). Observed effects were further confirmed in adoptive transfer of either total population of CS effector cells [lymph node plus spleen (LN + Spl)] or isolated T effector lymphocytes (T cells) that were treated with TNP-specific exosomes in the presence or absence of peritoneal or splenic macrophages from PCL-immunized donors (d). (a) Lack of suppression of active CS reaction in tolerized mice depleted of macrophages by intravenous or intraperitoneal administration of clodronate liposomes (groups C and E versus B, D and F). (b) Not suppressed adoptively transferred CS effector cells incubated with ultracentrifuged supernatant from culture of lymph node and spleen cells from tolerized mice depleted of macrophages (groups C and E versus B, D and F). (c) CS effector cells depleted of macrophages are not suppressed by TNP-specific exosomes (group D versus C) in contrast to total population of CS effector cells containing macrophages (group B versus A). (d) Isolated T effector lymphocytes are suppressed by Ts cell-derived exosomes only in the presence of splenic or peritoneal macrophages (groups H and J versus G and I). Total population of CS effector cells, which originally contains macrophages, is also suppressed by Ts cell exosomes (group B versus A). *P < 0·05; **P < 0·01; ***P < 0·001.

Exosome suppressive activity was assessed so far by the adoptive transfer of non-separated lymph node and spleen CS effector cells containing Mφ.4 To confirm the requirement for Mφ in this suppression mechanism, the adoptive transfer of CS effector cells depleted of Mφ and treated with exosomes or of isolated T effector lymphocytes treated with suppressive exosomes in the presence or absence of Mφ was performed. Interestingly, adoptively transferred CS effector cells depleted of Mφ were not suppressed by Ts cell-derived TNP-specific exosomes (Fig.2c, group D versus C). Furthermore, the suppression of the CS reaction was observed only in recipients of isolated T effector lymphocytes treated with exosomes in the presence of Mφ (Fig.2d, groups H and J) or exosome-treated CS effector cells originally containing Mφ (Fig.2d, group B), whereas direct treatment of isolated T effector lymphocytes with assayed exosomes in the absence of Mφ did not affect the transferred CS reaction (Fig.2d, group F). However, a CS reaction transferred by total effector cell mixture with additional Mφ (Fig.2d, group D) was suppressed by exosome treatment after 48 hr (data not shown). It should be stressed that after 48 hr, suppression of the CS reaction was still observed in groups H and J recipients. Both splenic and peritoneal Mφ from PCL-immunized donors were able to mediate the suppression, suggesting that the regulatory activity of Mφ is independent of their tissue origin.

Exosome-pulsed, hapten-labelled Mφ mediate the suppression of the CS response

Macrophages conjugated in vitro with hapten when transferred intravenously into naive recipients express reduced immunogenicity and induce the CS reaction in a significantly inferior manner compared with the response mediated by transferred lymph node and spleen CS effector cells.26 This effect could be affected by treatment of Mφ donors and/or recipients with a low dose of cyclophosphamide,27,28 which results in depletion of Treg cells.29,30 To investigate whether TNP-specific suppressive exosomes influence Mφ immunogenicity in the CS induction phase, peritoneal Mφ from naive mice were pre-treated with assayed exosomes, conjugated with TNP hapten (TNP-Mφ) and then transferred intravenously into either naive or cyclophosphamide-treated recipients, in which a CS reaction was elicited 7 days later. Pre-treatment of Mφ with suppressive exosomes did not affect the reduced CS reaction in naive recipients (Fig.3, group F versus E). Interestingly, transfer of exosome-pulsed TNP-Mφ resulted in a significantly inhibited CS reaction in cyclophosphamide-treated recipients (Fig.3, group C versus B). Hence, Mφ treated with suppressive exosomes and labelled with hapten may possess the decreased ability to induce cellular allergic immune response, which may be associated with activation of Treg cells de novo.

Figure 3.

Figure 3

Open in a new tab

Treatment with tolerogenic exosomes reduces immunogenicity of macrophages carrying antigen. Exosome-treated or naive macrophages were labelled with trinitrophenyl (TNP) hapten and then injected intravenously into naive or cyclophosphamide pre-treated recipients. Contact sensitivity (CS) reaction induced by transferred TNP-macrophages was elicited 7 days later by ear challenge and measured 24 hr later as ear swelling response. TNP-specific exosome-pulsed macrophages after in vitro labelling with TNP hapten very weakly induced CS response in recipients pre-treated with cyclophosphamide (group C versus B). *P < 0·05; **P < 0·01; ***P < 0·001.

T regulatory lymphocytes are required for immune suppression mediated by exosomes

Apart from direct inhibition of T effector cell proliferation by induction of apoptosis,31 Mφ were shown to suppress the cellular immune response by activation of Treg lymphocytes in a reactive oxygen intermediate-dependent manner.32 Previously we have ruled out the participation of FoxP3+ Treg lymphocytes in the generation of Ts cell-derived suppressive exosomes during tolerogenesis.4 However, the potential involvement of Treg cells induced by Mφ was not defined. To test this assumption, DEREG mice depleted of FoxP3+ Treg cells by intravenous injections of diphtheria toxin were tolerized with TNP-MRBC and contact immunized with PCL before the elicitation of an active CS reaction. Intravenous administration of TNP-MRBC slightly enhanced the CS response in DEREG mice (Fig.4, group B), suggesting that, possibly activated by exosome-pulsed Mφ, Treg lymphocytes participate in the effector phase of the present tolerance mechanism, i.e. in the suppression of CS effector T-cell proliferation mediated by exosome-treated Mφ.

Figure 4.

Figure 4

Open in a new tab

Participation of T regulatory lymphocytes in exosome-mediated suppression of contact sensitivity (CS). DEREG mice depleted of FoxP3+ regulatory T (Treg) cells by diphtheria toxin injections were tolerized by trinitrophenyl-labelled mouse red blood cell (TNP-MRBC) intravenous administration followed by picryl chloride (PCL) contact immunization. Then CS reaction was elicited by ear challenge and measured 24 hr later as ear swelling response. Intravenous administration of hapten-labelled syngeneic erythrocytes fails to suppress active CS response in DEREG mice depleted of Treg cells (group B versus A).

Suppressive exosomes inhibit proliferation of CS effector cells in the presence of Mφ

To determine the possible mechanism of CS reaction suppression mediated by exosome-pulsed Mφ, direct and transmembrane culture of CS effector cells with Mφ in the presence of antigen and suppressive exosomes was performed with the assessment of effector cell proliferation by incorporation of [3H]thymidine. Significant inhibition of antigen-stimulated proliferation of CS effector cells was observed in the presence of TNP-specific and SHAM-F exosomes with the strongest effect in transmembrane culture with Mφ (Fig.5, groups D and E versus C and groups G and H versus F).

Figure 5.

Figure 5

Open in a new tab

Exosomes suppress antigen-stimulated proliferation of contact sensitivity (CS) effector cells in the presence of macrophages. Lymph node and spleen effector cells from picryl chloride (PCL) -sensitized mice were cultured with macrophages in direct contact or in transmembrane manner for 24 hr in the presence of trinitrophenyl (TNP)-specific or Sham factor (antigen-non-specific suppressor factor) (SHAM-F) exosomes. Antigen-stimulated proliferation of CS effector cells was assessed by cell incorporation of [3H]thymidine during next 18 hr of culture. TNP-specific and SHAM-F exosomes reduce incorporation of [3H]thymidine by antigen-stimulated CS effector cells cultured either in direct contact or in transmembrane manner with macrophages (groups D and E versus C and groups G and H versus F). **P < 0·01; ***P < 0·001.

Discussion

Previous studies suggested the involvement of Mφ in an antigen-specific tolerance mechanism mediated by Ts lymphocytes and their biologically active product termed T-cell suppressor factor (TsF).7–10 Incubation of Mφ with TsF-containing supernatant reduced their immunogenicity as antigen-presenting cells leading to inhibition of the induction of the cellular immune response.7 The release of secondary macrophage suppressor factor by TsF-treated Mφ was also reported.6,11,12 However, the exact nature of TsF remained unclear for almost 30 years because of the inadequacy of techniques available at that time. Recently, Bryniarski et al.1–4 identified TsF as miRNA-150 carried by exosomes coated with hapten-specific antibody light-chains. This initiated research defining the mechanism of exosomal miRNA-150 suppressive action.

Although assayed exosomes are typically harvested from the supernatants of total lymph node and spleen cell cultures from tolerized mice,1–4 their CD8+ Ts cell origin has been unequivocally proved.3,4 Depletion of CD3+ and CD8+ cells before cell culture by specific monoclonal antibodies and complement abolished the suppressive activity of ultracentrifuged supernatant pellet,3,4 whereas the supernatant pellet from cultures of positively isolated CD8+ cells from lymph nodes and spleens of tolerized mice was strongly suppressive.3

The studies described here were aimed to investigate the role of Mφ in the passage of regulatory signal mediated by Ts cell-derived exosomes to effector cells of cellular as well as humoral immune responses. Because of the broad tissue distribution of Mφ and their ability to migrate into inflammatory sites, these cells seemed to be promising mediators of the tolerance mechanism.

Delemarre et al.33 used a depletion assay to show the important and well established role of Mφ in induction of humoral immune response to TNP-SRBC. Hence, the possible ability of Ts cell-derived exosomes to influence the humoral immune response induced by exosome-treated, antigen-pulsed Mφ was explored, showing the significant decrease in the number of splenic B cells releasing antigen-specific antibodies (Fig.1a) and the alteration of the relation of serum titres of specific IgM to IgG antibodies (Fig.1b). The observed effects may be due to impaired antigen phagocytosis, processing and presentation to induce T helper type 2 and B2 cells by Mφ pulsed with miRNA-150-containing exosomes, which resulted in inhibition of humoral immunity maturation and immunoglobulin class switching. This is in accordance with Xiao et al.34 demonstrating the inhibition of B2 cell maturation mediated by miRNA-150, which targeted transcription factor c-Myb. Further, miRNA-150 was shown to block the proliferation of B-cell receptor antigen-stimulated B cells.35 Hence, the direct transmission of miRNA-150 by exosome-affected macrophages to suppress the humoral response could not be excluded. Previously no differences in serum titres of TNP-specific antibodies were reported in mice injected intravenously with TNP-conjugated MRBC to induce TsF-mediated tolerance.36 However, our studies demonstrated that direct treatment of Mφ with suppressive exosomes impairs their ability to induce humoral immune response. Interestingly, a similar suppressive effect was observed in recipients of Mφ treated with SHAM-F exosomes before pulsing with antigen (Fig.1a,b, group C). SHAM-F exosomes act analogously to TsF exosomes and carry miRNA-150 (K. Bryniarski, P.W. Askenase unpublished results). However, these exosomes are not equipped with an antigen-recognition system, which reduces the effectiveness of their biological activity in vivo.

Nevertheless, as shown here, SHAM-F exosomes, despite of the lack of specific antibody light-chain coating, could be suppressive because they contain regulatory miRNA-150; the same miRNA that is responsible for the immune suppression mediated by hapten-MRBC-induced Ts cell-derived exosomes. Additionally, intravenous boost of syngeneic MRBC probably induces natural mechanisms of haematopoiesis regulation, including those mediated by miRNA-150. Therefore, as in both hapten-specific and SHAM-F exosomes, Ts cells are activated by intravenous application of syngeneic MRBC, the resulting miRNA-150-containing exosomes may originally function as regulators of haematopoiesis and then as immune suppressors, especially after coating with specific antibody light-chains.

Our current studies demonstrated that mice that were depleted in vivo of Mφ by administration of clodronate liposomes15,16 did not develop the suppression of CS reaction after tolerance induction (Fig.2a). Additionally, supernatant pellets from culture of Ts cells from mice depleted of Mφ before tolerization did not contain biologically active suppressive exosomes capable of inhibiting the transferred CS response (Fig.2b). Furthermore, CS effector cells depleted of Mφ from donors pre-treated with clodronate liposomes were not suppressed by Ts cell exosomes when transferred into naive recipients (Fig.2c). Moreover, CS reaction transferred by nylon wool-isolated T effector lymphocytes was only suppressed when T effector cells were treated with Ts cell-derived exosomes in the presence of splenic or peritoneal Mφ (Fig.2d). Notably, the CS effector cell populations obtained from lymph nodes and spleens that were used in all previous studies also originally contained Mφ, which were possibly responsible for the observed suppressive effect of exosome action.4 Hence, our observations confirmed the crucial role of Mφ in activation of Ts cells, as mentioned previously,37 as well as in the transmission of suppressive signal mediated by hapten-specific exosomes in hapten-induced allergic cellular immune responses. In addition, influence of exosomes on Mφ activity is not dependent on the tissue origin of these cells.

Intravenously transferred hapten-conjugated Mφ very weakly induce a CS reaction, leading to tolerance development7,26 that could be diminished by pre-treatment with a low dose of cyclophosphamide, which affects Ts38 and Treg cells.29,30 In the present experiments we have shown that exosome treatment did not influence TNP-labelled Mφ tolerogenic capability when cells were transferred into naive recipients (Fig.3). Interestingly, intravenously transferred exosome-treated TNP-Mφ induced significantly lower CS reaction in low-dose cyclophosphamide-treated recipients, whereas untreated TNP-Mφ were more immunogenic than tolerogenic (Fig.3), similarly to Mφ from mice lacking natural CD8+ Treg lymphocytes.39 It was concluded that the observed CS inhibition could be due to de novo induction of Treg lymphocytes by exosome-treated TNP-Mφ transferred into recipients depleted of Treg cells by pre-treatment with the low-dose cyclophosphamide.29,30 The exact mechanism of cellular interactions remains the subject of our ongoing studies. Flood et al.40 reported that pre-treatment of recipients with a low dose of cyclophosphamide abolished the suppression of CS transferred by effector cells incubated with TNP-specific TsF-containing supernatant. It was concluded that TsF-dependent suppression is mediated by cells sensitive to low doses of cyclophosphamide, which were recently identified as tolerogenic Mφ (resulting in enhancement of their immunogenicity)27,41 and Treg cells.29,30 However, the participation of FoxP3+ Treg lymphocytes in the generation of suppressive Ts cell-derived exosomes was recently excluded,4 because DEREG mice with depleted Treg cells13 produced functionally active Ts cell exosomes after tolerization.4 Hence, it was proposed that Treg cells are involved in the effector phase of studied tolerance mechanism and interact with Mφ as recently reviewed in other systems.42 To test this assumption, DEREG mice depleted of FoxP3+ Treg lymphocytes were injected with TNP-MRBC before the induction of active CS by skin application of hapten followed by ear challenge and measurement of CS. Tolerized and then actively sensitized DEREG mice developed an unaffected CS reaction (Fig.4), which confirmed the involvement of Treg lymphocytes in the effector mechanism of tolerance mediated by Ts cell-derived exosomes acting through Mφ.

Further, significant inhibition of antigen-stimulated proliferation of CS effector cells was shown under the influence of Ts cell-derived exosomes added to effector cells cultured with Mφ, either in direct contact or in transmembrane manner (Fig.5). These observations suggest the ability of exosome-treated Mφ to inhibit differentiation of T effector lymphocytes possibly mediated by soluble factor. Our preliminary results also suggest the induction of apoptosis of CS effector T lymphocytes treated with exosomes in the presence of Mφ, which could be induced, as recently reported,43 by miRNA-150 carried by assayed exosomes of TNP-specific TsF and SHAM-F origin. This suggests the transmission of miRNA-150 by exosome-treated Mφ to effector T lymphocytes possibly through secondary exosomes, that resemble macrophage suppressor factor,6,11,12 according to our preliminary data.44

Moreover, it was shown recently that, apart from the significant enhancement in generation of reactive oxygen intermediates by exosome-treated Mφ, their secretory activity assayed as release of cytokines and nitric oxide as well as the expression of selected surface markers of antigen phagocytosis and presentation remain unaffected under the influence of Ts cell-derived exosomes,45 proving the specificity of the tested tolerance mechanism. Furthermore, macrophage-derived reactive oxygen intermediates were shown to both activate the apoptosis of differentiated T effector cells31 and induce Treg cells,32,46 hence these radicals may possibly enhance the efficiency of exosome-treated Mφ suppressive activity.

To summarize, the present data describe the mechanism of antigen-specific immune tolerance mediated by Ts cell-derived exosomes carrying miRNA-150, the generation and suppressive activity of which is dependent on Mφ acting through the induction of Treg cells and the inhibition of proliferation of T effector lymphocytes. To our knowledge, the essential role of Mφ in antigen-specific immune suppression mediated by exosomes carrying immunoregulatory miRNA was shown here for the first time.

Contributions to authorship

KN performed the experiments, analysed the data and wrote the manuscript. WP measured some of the CS ear swellings, consulted experimental protocols and revised the manuscript. BN conducted the radioactivity measurement in the proliferation assay. MP supported the preparation of cell cultures. PWA supported experiments with DEREG mice, discussed experimental protocols and revised the manuscript. KB measured some of the CS ear swellings, consulted and assisted in performance of experiments, revised the manuscript and supervised co-workers.

Acknowledgments

The authors thank Harumichi Ishigame (Yale University School of Medicine) for cytofluorometric evaluation of Treg cell content in provided samples collected from DEREG mice. This study was supported by a Polish National Science Centre grant (No. 2013/09/N/NZ6/00753) to KN. Ultracentrifuge purchase was supported by the Polish Ministry of Science and Higher Education (funding No. 6354/IA/156/2013).

Glossary

CS

contact sensitivity

KO

knock-out

MGG

mouse γ-globulins

miRNA

microRNA

Mφ

macrophages

NF

negative factor (for control of exosomes)

PCL

picryl chloride (trinitrophenyl chloride)

SHAM-F

Sham factor (antigen-non-specific suppressor factor)

SRBC

sheep red blood cells

TNBSA

trinitrobenzene sulphonic acid

TNP

trinitrophenol (hapten)

TNP-Mφ

trinitrophenyl-conjugated macrophages

TNP-MRBC

trinitrophenyl-conjugated murine red blood cells

Treg cells

T regulatory lymphocytes

Ts cells

T CD8+ suppressor lymphocytes

TsF

T suppressor factor (antigen-specific)

Disclosures

The authors declare no financial or commercial conflict of interests.

References

  1. Bryniarski K, Nazimek K, Martin E, Ptak M, Askenase PW, Ptak W. T CD8+ cell-derived exosomal miRNA-150 suppresses induction and effector phases of murine contact sensitivity as well as symptoms of active allergy. Cent Eur J Immunol. 2014;39(Suppl. 1):20. [Google Scholar]
  2. Bryniarski K, Nazimek K, Sikora E, Nowak B, Ptak M, Askenase PW, Ptak W. miRNA-150 inhibits contact sensitivity response via antigen-specific exosomes produced by T suppressor cells. Immunology. 2012;137(Suppl. 1):91. [Google Scholar]
  3. Bryniarski K, Nazimek K, Sikora E, Ptak M, Askenase PW, Ptak W. TCD8+ suppressor cells produce antigen-specific exosomes carrying miRNA-150 to inhibit contact sensitivity response. Front Immunol. 2013 doi: 10.3389/conf.fimmu.2013.02.00077. [Conference Abstract: 15th International Congress of Immunology (ICI)] [Google Scholar]
  4. Bryniarski K, Ptak W, Jayakumar A, et al. Antigen specific, antibody coated, exosome-like nanovesicles deliver suppressor T cell miRNA-150 to effector T cells in contact sensitivity. J Allergy Clin Immunol. 2013;132:170–81. doi: 10.1016/j.jaci.2013.04.048. e9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Rosenstein RW, Murray JH, Cone RE, Ptak W, Iverson GM, Gershon RK. Isolation and partial characterization of an antigen-specific T-cell factor associated with the suppression of delayed type hypersensitivity. Proc Natl Acad Sci USA. 1981;78:5821–5. doi: 10.1073/pnas.78.9.5821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Marcinkiewicz J, Ptak W. Macrophage suppressor factor in contact sensitivity. Mechanisms of its release and action. Immunology. 1980;41:211–6. [PMC free article] [PubMed] [Google Scholar]
  7. Ptak W, Gershon RK. Immunological agnosis: a state that derives from T suppressor cell inhibition of antigen presenting cells. Proc Natl Acad Sci USA. 1982;79:2645–8. doi: 10.1073/pnas.79.8.2645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ptak W, Marcinkiewicz J, Rewicka M, Rozycka D. The requirement for T lymphocyte-specific suppressor factors and corresponding antigen in the production of nonspecific suppressor monokines by macrophages. J Reticuloendothel Soc. 1980;27:575–84. [PubMed] [Google Scholar]
  9. Ptak W, Naidorf KF, Gershon RK. Interference with the transmission of T-cell derived messages by macrophage membranes. J Immunol. 1977;119:444–9. [PubMed] [Google Scholar]
  10. Ptak W, Zembala M, Asherson GL, Marcinkiewicz J. Inhibition of contact sensitivity by macrophages. Int Arch Allergy Appl Immunol. 1981;65:121–8. doi: 10.1159/000232747. [DOI] [PubMed] [Google Scholar]
  11. Ptak W, Zembala M, Gershon RK. Intermediary role of macrophages in the passage of suppressor signals between T-cell subsets. J Exp Med. 1978;148:424–34. doi: 10.1084/jem.148.2.424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ptak W, Zembala M, Hanczakowski-Rewicka M, Asherson GL. Nonspecific macrophage suppressor factor: its role in the inhibition of contact sensitivity to picryl chloride by specific T suppressor factor. Eur J Immunol. 1978;8:645–9. doi: 10.1002/eji.1830080908. [DOI] [PubMed] [Google Scholar]
  13. Lahl K, Sparwasser T. In vivo depletion of FoxP3+ Tregs using the DEREG mouse model. Methods Mol Biol. 2011;707:157–72. doi: 10.1007/978-1-61737-979-6_10. [DOI] [PubMed] [Google Scholar]
  14. Ptak W, Szczepanik M, Bryniarski K, Tutaj M, Ptak M. Epicutaneous application of protein antigens incorporated into cosmetic cream induces antigen-nonspecific unresponsiveness in mice and affects the cell-mediated immune response. Int Arch Allergy Immunol. 2002;128:8–14. doi: 10.1159/000057998. [DOI] [PubMed] [Google Scholar]
  15. Van Rooijen N, Sanders A. Elimination, blocking, and activation of macrophages: three of a kind? J Leukoc Biol. 1997;62:702–9. doi: 10.1002/jlb.62.6.702. [DOI] [PubMed] [Google Scholar]
  16. Van Rooijen N, Sanders A. Liposome mediated depletion of macrophages: mechanism of action, preparation of liposomes and applications. J Immunol Methods. 1994;174:83–93. doi: 10.1016/0022-1759(94)90012-4. [DOI] [PubMed] [Google Scholar]
  17. Ptak W, Rozycka D, Rewicka M. Induction of suppressor cells and cells producing antigen-specific suppressor factors by haptens bound to self carriers. Immunobiology. 1980;156:400–9. doi: 10.1016/S0171-2985(80)80073-8. [DOI] [PubMed] [Google Scholar]
  18. Filipczak-Bryniarska I, Nowak B, Sikora E, Nazimek K, Woron J, Wordliczek J, Bryniarski K. The influence of opioids on the humoral and cell-mediated immune responses in mice. The role of macrophages. Pharmacol Rep. 2012;64:1200–15. doi: 10.1016/s1734-1140(12)70916-7. [DOI] [PubMed] [Google Scholar]
  19. Czajkowska B, Ptak M, Bobek M, Bryniarski K, Szczepanik M. Different isoenzyme patterns of nonspecific esterases and the level of IL-6 production as markers of macrophage functions. Folia Histochem Cytobiol. 1995;33:111–5. [PubMed] [Google Scholar]
  20. Asherson GL, Ptak W. Contact and delayed hypersensitivity in the mouse. I. Active sensitization and passive transfer. Immunology. 1968;15:405–16. [PMC free article] [PubMed] [Google Scholar]
  21. Zemelka-Wiacek M, Majewska-Szczepanik M, Pyrczak W, Szczepanik M. Complementary methods for contact hypersensitivity (CHS) evaluation in mice. J Immunol Methods. 2013;387:270–5. doi: 10.1016/j.jim.2012.11.004. [DOI] [PubMed] [Google Scholar]
  22. Kokkinopoulos D, Perez S, Sotiriadou R, Stinios J, Papamichail M. The use of nylon wool for the isolation of T lymphocyte subpopulations. J Immunol Methods. 1992;154:1–6. doi: 10.1016/0022-1759(92)90205-8. [DOI] [PubMed] [Google Scholar]
  23. Bryniarski K, Szczepanik M, Maresz K, Ptak M, Ptak W. Subpopulations of mouse testicular macrophages and their immunoregulatory function. Am J Reprod Immunol. 2004;52:27–35. doi: 10.1111/j.1600-0897.2004.00178.x. [DOI] [PubMed] [Google Scholar]
  24. Nazimek K, Ptak M, Ptak W, Bryniarski K. T CD8+ suppressor cell-derived exosomes carrying miRNA-150 impair macrophage ability to induce humoral immune response in mice. Front Immunol. 2013 doi: 10.3389/conf.fimmu.2013.02.00023. [Conference Abstract: 15th International Congress of Immunology (ICI)] [Google Scholar]
  25. Wilson SD, Munson AE, Meade BJ. Assessment of the functional integrity of the humoral immune response: the plaque-forming cell assay and the enzyme-linked immunosorbent assay. Methods. 1999;19:3–7. doi: 10.1006/meth.1999.0821. [DOI] [PubMed] [Google Scholar]
  26. Ptak W, Bereta M, Ptak M, Iverson GM, Green DR. Suppression and contrasuppression in the induction of contact sensitivity by the administration of cell bound antigen–antibody complexes. J Immunol. 1985;135:2312–8. [PubMed] [Google Scholar]
  27. Bryniarski K, Szczepanik M, Ptak M, Zemelka M, Ptak W. Influence of cyclophosphamide and its metabolic products on the activity of peritoneal macrophages in mice. Pharmacol Rep. 2009;61:550–7. doi: 10.1016/s1734-1140(09)70098-2. [DOI] [PubMed] [Google Scholar]
  28. Nazimek K, Sikora E, Nowak B, Ptak M, Ptak W, Bryniarski K. Low dose of cyclophosphamide alleviates the suppression of contact sensitivity response mediated by macrophages. Immunology. 2012;137(Suppl. 1):696–7. [Google Scholar]
  29. Ghiringhelli F, Larmonier N, Schmitt E, et al. CD4+ CD25+ regulatory T cells suppress tumor immunity but are sensitive to cyclophosphamide which allows immunotherapy of established tumors to be curative. Eur J Immunol. 2004;34:336–44. doi: 10.1002/eji.200324181. [DOI] [PubMed] [Google Scholar]
  30. Zhao J, Cao Y, Lei Z, Yang Z, Zhang B, Huang B. Selective depletion of CD4+ CD25+ Foxp3+ regulatory T cells by low-dose cyclophosphamide is explained by reduced intracellular ATP levels. Cancer Res. 2010;70:4850–8. doi: 10.1158/0008-5472.CAN-10-0283. [DOI] [PubMed] [Google Scholar]
  31. Tripathi P, Hilderman D. Sensitization of T cells to apoptosis – a role for ROS? Apoptosis. 2004;9:515–23. doi: 10.1023/B:APPT.0000038033.14925.02. [DOI] [PubMed] [Google Scholar]
  32. Kraaij MD, Savage ND, van der Kooij SW, et al. Induction of regulatory T cells by macrophages is dependent on production of reactive oxygen species. Proc Natl Acad Sci USA. 2010;107:17686–91. doi: 10.1073/pnas.1012016107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Delemarre FG, Kors N, van Rooijen N. Elimination of spleen and lymph node macrophages and its difference in the effect on the immune response to particulate antigens. Immunobiology. 1990;182:70–8. doi: 10.1016/S0171-2985(11)80584-X. [DOI] [PubMed] [Google Scholar]
  34. Xiao C, Calado DP, Galler G, et al. MiR-150 controls B cell differentiation by targeting the transcription factor c-Myb. Cell. 2007;131:146–59. doi: 10.1016/j.cell.2007.07.021. [DOI] [PubMed] [Google Scholar]
  35. Kluiver JL, Chen CZ. MicroRNAs regulate B-cell receptor signaling-induced apoptosis. Genes Immun. 2012;13:239–44. doi: 10.1038/gene.2012.1. [DOI] [PubMed] [Google Scholar]
  36. Ptak W, Rozycka D. Split unresponsiveness to the trinitrophenyl determinant. I. Manoeuvers which suppress either humoral or cell-mediated immune responses. Eur J Immunol. 1977;7:855–60. doi: 10.1002/eji.1830071207. [DOI] [PubMed] [Google Scholar]
  37. Usui M, Aoki I, Sunshine GH, Dorf ME. A role for macrophages in suppressor cell induction. J Immunol. 1984;132:1728–34. [PubMed] [Google Scholar]
  38. Askenase PW, Hayden BJ, Gershon RK. Augmentation of delayed-type hypersensitivity by doses of cyclophosphamide which do not affect antibody responses. J Exp Med. 1975;141:697–702. doi: 10.1084/jem.141.3.697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Bryniarski K, Szczepanik M, Ptak M, Sikora E, Majewska-Szczepanik M, Ptak W. Down-regulation of macrophage immune activity by natural CD8+ regulatory T cells. Folia Biol. 2013;61:65–72. doi: 10.3409/fb61_1-2.65. [DOI] [PubMed] [Google Scholar]
  40. Flood P, Ptak W, Green DR. Mechanism of action of a T suppressor factor (TsF) in contact sensitivity: the T cell target for TsF activity in adoptive transfer of immunity is not effector cell. J Immunol. 1986;137:1829–35. [PubMed] [Google Scholar]
  41. Marcinkiewicz J, Bryniarski K, Ptak W. Cyclophosphamide uncovers two separate macrophage subpopulations with opposite immunogenic potential and different patterns of monokine production. Cytokine. 1994;6:472–7. doi: 10.1016/1043-4666(94)90073-6. [DOI] [PubMed] [Google Scholar]
  42. Zhen Y, Zheng J, Zhao Y. Regulatory CD4+ CD25+ T cells and macrophages: communication between two regulators of effector T cells. Inflamm Res. 2008;57:564–70. doi: 10.1007/s00011-008-7234-3. [DOI] [PubMed] [Google Scholar]
  43. Ghisi M, Corradin A, Basso K, et al. Modulation of microRNA expression in human T-cell development: targeting of NOTCH3 by miR-150. Blood. 2011;117:7053–62. doi: 10.1182/blood-2010-12-326629. [DOI] [PubMed] [Google Scholar]
  44. Nazimek K, Nowak B, Ptak M, Ptak W, Bryniarski K. Essential role of macrophages in antigen-specific suppression of immune response mediated by T CD8+ lymphocyte-derived regulatory exosomes. Cent Eur J Immunol. 2014;39(Suppl. 1):10. [Google Scholar]
  45. Nazimek K, Nowak B, Marcinkiewicz J, Ptak M, Ptak W, Bryniarski K. Enhanced generation of reactive oxygen intermediates by suppressor T cell-derived exosome-treated macrophages. Folia Med Cracov. 2014;54:37–52. [PubMed] [Google Scholar]
  46. Gelderman KA, Hultqvist M, Pizzolla A, Zhao M, Nandakumar KS, Mattsson R, Holmdahl R. Macrophages suppress T cell responses and arthritis development in mice by producing reactive oxygen species. J Clin Invest. 2007;117:3020–8. doi: 10.1172/JCI31935. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Immunology are provided here courtesy of British Society for Immunology

RESOURCES