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The Journal of Immunology, 2006, 177: 2691-2698.
Copyright © 2006 by The American Association of Immunologists

Monocyte-Derived Human Macrophages Mediate Anergy in Allogeneic T Cells and Induce Regulatory T Cells1

Sabine Hoves*, Stefan W. Krause{dagger}, Christian Schütz*, Dagmar Halbritter*, Jürgen Schölmerich*, Hans Herfarth* and Martin Fleck2,*

* Department of Internal Medicine I, University of Regensburg, Regensburg, Germany; and {dagger} Division of Hematology and Oncology, Department of Internal Medicine I, University of Regensburg, Regensburg, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Activation of alloreactive T cells by APCs such as dendritic cells (DC) has been implicated as crucial step in transplant rejection. In contrast, it has been proposed that macrophages (M{phi}) maintain tolerance toward alloantigens. It was therefore the aim of this study to further analyze the T cell-stimulatory capacity of mature DC and M{phi} in vitro using the model of allogeneic MLR. There was a strong proliferative response in T cells cocultured with DC, which was further increased upon restimulation in a secondary MLR. In contrast, T cells did not proliferate in cocultures with M{phi} despite costimulation with anti-CD28 and IL-2. Cytokine analysis revealed considerable levels of IL-10 in cocultures of T cells with M{phi}, whereas high amounts of IL-2 and IFN-{gamma} were present in cocultures with DC. There was only minimal T cell proliferation in a secondary MLR when T cells were rescued from primary MLR with M{phi} and restimulated with DC of the same donor, or DC of an unrelated donor (third party), whereas a strong primary proliferative response was observed in resting T cells, demonstrating induction of T cell anergy by M{phi}. Functional analysis of T cells rescued from cocultures with M{phi} demonstrated that anergy was at least partly mediated by IL-10-producing regulatory T cells induced by M{phi}. These results demonstrate that M{phi} drive the differentiation of regulatory T cells and mediate anergy in allogeneic T cells, supporting the concept that M{phi} maintain peripheral tolerance in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In the past decade, much attention has been focused on dendritic cells (DC),3 which are potent APCs, and there are numerous studies demonstrating the exceptional potency of DC to induce primary and secondary T cell responses (1). This process requires terminal maturation of DC including activation via TLR and TNFR family members leading to enhanced Ag presentation and up-regulated expression of costimulatory molecules. In contrast to mature DC (mDC), immature or semimature DC have a distinct role in regulating immune responses and increasing evidence suggests that these cells promote tolerance rather than immunity (2, 3, 4).

As DC, macrophages (M{phi}) differentiate from myeloid precursors and form a heterogeneous population of APC that link the innate and adaptive immune systems (5). Three types of activated M{phi} have been characterized which exhibit different phenotypes and perform distinct immunological functions. Classical activation of M{phi} requires priming by IFN-{gamma} followed by exposure to TNF or a TNF inducer like bacteria or bacterial products such as LPS. These classically activated M{phi} possess a markedly enhanced ability to kill and degrade intracellular microorganisms and support specific T cell responses by Ag presentation and release of several cytokines, including IL-12 (5, 6). An alternative activation phenotype has been generated by stimulation of M{phi} with IL-4, resulting in production of IL-10 and IL-1R antagonist (7, 8). These alternatively activated M{phi} are not efficient at Ag presentation and could suppress proliferation of mitogen-activated T cells in vitro. However, these M{phi} produce high levels of matrix-associated proteins and promote fibrogenesis, suggesting a role in tissue repair and wound healing (9). Exposure of M{phi} to classical activating signals in the presence of IgG-containing immune complexes resulted in a cell type different from classically or alternatively activated M{phi}. These type II activated M{phi} produce high levels of IL-10 upon Fc{gamma}R ligation and activation via TLRs as well as through CD40 or CD44, resulting in potent anti-inflammatory effects and IL-4 induction in activated T cells (5, 10).

In contrast with activated M{phi} populations, the biological functions of resting M{phi} are not well defined. Because resting M{phi} possess nearly the same phagocytic activity as activated M{phi}, it appears to be the major task of these cells to dispose apoptotic cells and cell debris (11). However, M{phi} have also been implicated in suppression of autoreactive T cells, suggesting an active role in the immune system by maintaining tolerance toward self Ags (12, 13, 14, 15).

To further evaluate the T cell-stimulatory capacity of resting M{phi} in comparison with mDC, coculture experiments with allogeneic T cells were performed using the model of allogeneic MLR. The present results demonstrate that M{phi} mediate anergy in T cells, which is at least partly dependent on regulatory T cells induced by M{phi}, supporting the concept that resting M{phi} maintain peripheral tolerance of T cells in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Preparation and culture of M{phi}, DC, and T cells

PBMC (mononuclear cells (MNC)) were isolated from leukapheresis concentrates of healthy donors by density gradient centrifugation. Monocytes were separated from MNC by countercurrent elutriation in a J6M-E Beckman centrifuge with a large chamber and a JE-5 rotor at 1100 x g at a flow rate of 110 ml/min in HBSS as described previously (16). Elutriated monocytes were >90% pure as determined by morphology and Ag expression (CD14, CD3, CD20) measured by flow cytometry.

To induce the in vitro differentiation of monocytes to M{phi}, monocytes were cultured in Teflon bags in RPMI 1640 culture medium (BioWhittaker Europe) supplemented with 200 mM L-glutamine, 100 mM sodium pyruvate, nonessential amino acids, minimal essential medium vitamins (all from Invitrogen Life Technologies), penicillin/streptomycin (50 U/50 µg/ml; PAA Laboratories) and 50 µM mercaptoethanol (Amresco) in the presence of 2% human AB serum as previously described (17). For functional assays and phenotypical analysis, M{phi} were harvested on day 5.

To induce the in vitro differentiation of monocytes to DC, monocytes were cultured for 7 days in serum-free CellGro culture medium (CellGenix) in the presence of 500 U/ml IL-4 (Promocell) and 500 U/ml GM-CSF (Leukomax; Essex). For maturation of DC, cells were additionally stimulated with IL-1beta (10 ng/ml), TNF-{alpha} (10 ng/ml), IL-6 (1000 U/ml; all from Promocell), and PGE2 (1 µg/ml; Minprostin E2; Pharmacia and Upjohn) for an additional 2 days as described previously (18).

T cells were also separated from MNC by countercurrent elutriation and frozen immediately in RPMI culture medium containing 10% DMSO (Sigma-Aldrich) and 40% heat-inactivated (56°C for 30 min) autologous plasma. For additional experiments, allogeneic T cells were rapidly thawed, washed with PBS (Life Technologies), and resuspended in RPMI 1640 culture medium with 5% autologous plasma.

FACS

Surface markers expressed by monocyte-derived M{phi} and DC were determined by flow cytometry using the following mAbs: FITC-conjugated anti-CD80 (clone B7.5), PE-conjugated anti-CD86 (clone VI CD86.8), and matching isotype control Abs (all purchased from BD Pharmingen); PE-conjugated anti-CD83 (clone HB15As; Immunotech); and FITC-conjugated anti-HLA-DR (clone B-F1s; Trinova Biochem).

After one washing with PBS containing 10% FCS, cells were incubated with mAbs or isotype control Abs for 30 min. After washing twice with PBS, cells were fixed in PBS containing 1% paraformaldehyde (Sigma-Aldrich), and 10,000 gated events were analyzed using an EPICSXL-MCL (Coulter Electronics).

Analysis of T cell surface markers were performed on a FACSCalibur using the following mAbs: FITC- or PE-conjugated anti-CD3 (clone SK7), PE- or PerCP-conjugated anti-CD4 (clone SK3), PerCP- or allophycocyanin-conjugated CD8 (clone SK1), FITC-conjugated anti-CD69 (clone L78, all obtained from BD Biosciences); anti-CCR7 (clone 150503; R&D Systems); as well as allophycocyanin-conjugated anti-CD25 (clone M-A251, BD Biosciences); anti-CD45 RA (clone MEM 56) and anti-CD45 RO (clone UCHL1, both from Caltag); and anti-CTLA-4 (clone BN 13; BD Biosciences).

Primary allogeneic MLR

For primary MLR, 5 x 104 T cells were incubated with different numbers of allogeneic M{phi} or mature DC (mDC) from the same donor at stimulator-responder ratios ranging from 1:625 to 1:1 in 96-well round-bottom tissue culture plates (Nunc) in a total volume of 200 µl of RPMI 1640 containing 5% T cell autologous plasma in a humidified incubator (37°C, 5% CO2). On day 5 of coculture, 1 µCi of [methyl-3H]thymidine/well (PerkinElmer) was added. After 20 h, labeled DNA of proliferating cells was harvested onto glass fiber filters (Printed Filtermat B; Wallac Oy) using a Vacusafe IH-280 harvester (Innotech). Filters were transferred to scintillation fluid (Betaplate Scint; Wallac), and incorporated radioactivity was determined by a liquid scintillation counter (1450 MicroBeta; Wallac). All samples were tested in triplicates, and values are indicated as means ± SD.

To analyze the hyporesponsivness of T cells in cocultures with M{phi}, IL-2 (100 U/ml; Promocell), anti-CD28 Ab (clone CD28.2; eBioscience), or a combination of both was added to the primary MLR. As control, plates were coated with anti-CD3 (clone OKT-3; eBioscience), and T cells were cultured in the presence of anti-CD28 and IL-2 as previously described (19, 20).

Cytokine-ELISA

For determination of cytokine production, cocultures of T cells with allogeneic M{phi} or mDC were established in 24-well plates. Every day, one well was harvested, and the supernatant was preserved for further analysis. Amounts of IFN-{gamma}, IL-10, and IL-2 were analyzed using specific ELISA kits (R&D Systems).

Restimulation in secondary MLR

For analysis of allospecific activation of T cells, restimulation experiments were performed. Primary MLR were established as bulk cultures in 48-well tissue culture plates (BD Biosciences) using 2 x 105 M{phi} or mDC from the same donor as stimulator cells and 106 allogeneic T cells per well as responder cells (stimulator-responder ratio, 1:5) in a total volume of 1 ml of RPMI medium containing 5% T cell autologous plasma. On day 6 of coculture, T cells were harvested from the primary MLR, and the number of living cells was determined by trypan blue exclusion (Sigma-Aldrich). In most experiments, aliquots of living T cells were immediately restimulated in a secondary MLR with mDC (stimulator-responder ratios ranging from 1:625 to 1:1) of the same donor as used in the primary MLR, or with mDC of a different donor (third party). Proliferation of T cells was determined by [methyl-3H]thymidine incorporation after 48 h of coculture. In additional experiments, T cells were rescued from primary cocultures and propagated during a resting period in RPMI 1640 culture medium containing 5% T cell autologous plasma with or without the presence of low dose IL-2 (10 U/ml) for 5 days. Afterwards, T cells were restimulated for 72 h as described previously. Cocultures were performed in triplicates, and values are indicated as means ± SD.

Anergy induction of preactivated T cells

To analyze the potential of M{phi} to induce anergy in preactivated T cells, multiple rounds of stimulation with T cells were performed. Primary MLR cultures were established in 48-well plates at a stimulator-responder ratio of 1:5 for 6 days with mDC and allogeneic T cells. To determine the proliferative response, cocultures were also established in 96-well plates as described above (stimulator-responder ratios from 1:625 to 1:1) and [methyl-3H]thymidine incorporation was analyzed on the day the bulk cultures were harvested. T cells were rescued from bulk cultures and stimulated for 72 h in a secondary MLR with M{phi} generated from the same donor of the mDC during the primary MLR. T cells were rescued again from the secondary MLR bulk cultures, and T cell proliferation was determined. In addition, aliquots of rescued T cells were restimulated with mDC of the same donor in a third MLR. Proliferation of T cells was determined after 48 h by incorporation of [methyl-3H]thymidine.

Suppression assay

T cells were stimulated in a primary MLR with allogeneic M{phi} in bulk cultures for 6 days as described above. T cells were rescued, and 50,000 T cells were added to cocultures of 50,000 autologous resting T cells with different numbers of allogeneic mDC at stimulator-responder ratios ranging from 1:625 to 1:1. Control cultures were established with mDC, and 50,000 or 100,000 resting T cells or T cells were rescued from cocultures with M{phi}. Proliferation of T cells was determined after 6 days by [methyl-3H]thymidine incorporation. All samples were tested in triplicates, and values are indicated as means ± SD.

Cell number-dependent suppression was analyzed by addition of increasing numbers (10,000–200,000) of regulatory T cells derived from M{phi} cocultures to 50,000 autologous T cells; 10,000 allogeneic mDC served as stimulatory cells. Proliferation of T cells was determined after 6 days by [methyl-3H]thymidine incorporation.

RT-PCR

CD4+ and CD8+ T cells from cocultures with allogeneic M{phi} or mDC were enriched by positive selection using MACS Beads (Miltenyi Biotec). Total RNA was isolated from these cells using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. Total RNA (100 ng) was used to synthesize cDNA using the First Strand cDNA Synthesis Kit, including avian myeloblastosis virus for reverse transcription (Roche Diagnostics). RT-PCR for human IL-10 was performed using the forward primer 5'-CAGTCTGAGAACAGCTGCAC-3' and the reverse primer 5'-TCACATGCGCCTTGATGTCT-3' (product, 255 bp). Primers for amplification of 18S mRNA were forward 5'-TCAAGAACGAAAGTCGGAG-3' and reverse 5'-GGACATCTAAGGGCATCACA-3' (product, 488 bp). RT-PCR was performed using the TaqPCR Master Mix Kit (Qiagen), 10 pmol from each primer of a primer pair and 2 µl of cDNA. All samples were incubated for 30 cycles using a thermocycler (GeneAmp PCR System 9700; PE Applied Biosystems). RT-PCR products were separated on a 1% agarose gel and visualized by GelStar staining (BMA Products).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Phenotype of M{phi} and DC differentiated in vitro from primary human monocytes

M{phi}, immature DC, and mDC were generated as described above. At day 7 of culture, the phenotypes of M{phi} and DC were determined by expression of characteristic surface markers using FACS. Consistent with previous reports (21), DC generated from monocytes in the presence of IL-4 and GM-CSF expressed considerable levels of HLA class II and low levels of costimulatory molecules CD80 and CD86, whereas CD83 was almost undetectable, confirming the immature phenotype. Upon stimulation with IL-1beta, TNF-{alpha}, IL-6, and PGE2, DC exhibited a fully mature phenotype as demonstrated by increased expression of HLA class II, CD80, CD86, and CD83. In contrast, low levels of costimulatory molecules could be detected on the surface of monocyte-derived M{phi} (Fig. 1). For additional experiments, cocultures of T cells were established with mDC or M{phi}.


Figure 1
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  		FIGURE 1. Primary human monocytes were differentiated in vitro into M{phi}, immature DC, and mDC. At day 7 of culture, aliquots of cells were obtained, and the phenotypes of M{phi} and DC were determined by expression of characteristic surface markers via FACS.

 
Proliferation of T cells in cocultures with allogeneic M{phi} or DC

To compare the stimulatory capacity, cocultures were established of mDC and M{phi} with allogeneic T cells, and proliferation of T cells was determined. Analysis of [methyl-3H]thymidine incorporation at day 6 revealed a strong proliferative response in T cells cocultured with mDC, whereas no proliferation could be observed in T cells cocultured with M{phi} (Fig. 2). Increasing levels of IFN-{gamma} and high concentrations of IL-2 with a maximal production at day 2 were present in cocultures of mDC and allogeneic T cells. In contrast, only low levels of of IL-2 as well as IFN-{gamma} and considerable amounts of IL-10 could be detected in cocultures of T cells with M{phi} (Fig. 3).


Figure 2
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  		FIGURE 2. Cocultures were established of mDC or M{phi} with allogeneic T cells at different stimulator-responder ratios, and proliferation of T cells was determined at day 6 using [methyl-3H]thymidine incorporation. Results are representative of seven independent experiments. Bars, SD.

 

Figure 3
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  		FIGURE 3. Cytokine production was analyzed in cocultures of mDC and M{phi} with allogeneic T cells at different time points using specific ELISAs. *, p < 0.05 (Mann-Whitney U test)

 
To investigate whether hyporesponsivness of T cells was due to the lack of costimulatory signals provided by the M{phi}, activating anti-CD28 mAbs with and without IL-2 were added to the cocultures of M{phi} with allogeneic T cells. However, T cell proliferation could be observed only at low levels in the presence of anti-CD28 mAbs and IL-2, suggesting that hyporesponsivness of T cells was actively induced by M{phi} and not a consequence of lack of costimulation (Fig. 4).


Figure 4
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  		FIGURE 4. Activating anti-CD28 mAbs with and without IL-2 were added to the cocultures of allogeneic T cells with M{phi}, and proliferation of T cells was determined at day 6 using [methyl-3H]thymidine incorporation. As a positive control, T cells were stimulated with anti-CD3, anti-CD28, and IL-2. One of three independent experiments is depicted.

 
Unresponsiveness of T cells cocultured with M{phi} was due to anergy induction

To further analyze whether the unresponsiveness observed in T cells cocultured with allogeneic M{phi} was due to anergy induction, T cells were rescued from primary cocultures with M{phi} at day 6 and immediately restimulated in a secondary MLR with mDC of the same donor from which the M{phi} had been generated (donor A), or an unrelated donor with a different haplotype (third-party donor B). There was no proliferative response in T cells stimulated by mDC in the secondary MLR, suggesting that T cells were rendered anergic during the first coculture with M{phi} (Fig. 5A). To formally demonstrate anergy, additional coculture experiments were established using autologous resting primary T cells as control cells. A strong proliferative T cell response could be observed in cocultures of resting primary T cells with allogeneic mDC, whereas proliferation was minimal in T cells rescued from primary cocultures with allogeneic M{phi} and immediately restimulated with mDC (Fig. 5B). These results demonstrated that unresponsiveness of T cells cocultured with M{phi} was due to induction of anergy.


Figure 5
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  		FIGURE 5. T cells were rescued from primary cocultures with M{phi} or mDC at day 6 and restimulated in a secondary MLR with different numbers of mDC of the same donor from which the primary stimulator cells had been generated (donor A) or an unrelated donor with a different haplotype (third- party donor B). Proliferation of T cells was determined at day 3 using [methyl-3H]thymidine incorporation (A). To formally demonstrate anergy, additional coculture experiments were established using resting primary T cells as control cells. Proliferation of T cells was determined at day 6 using [methyl-3H]thymidine incorporation (B). One of three to five independent experiments is depicted.

 
Anergy in T cells was reversed by exogenous IL-2 in the absence of M{phi}

To analyze whether anergy could be overcome by addition of IL-2 in the absence of M{phi}, T cells were rescued from primary cocultures with allogeneic M{phi} of donor A at day 6 and cultured with or without low-dose IL-2 for 5 days before restimulation in a secondary MLR with fully mature DC of donor A or DC of an unrelated donor B. Low-level T cell proliferation could already be observed in cultures of T cells without IL-2 treatment, suggesting that anergy in T cells required the presence of M{phi} (Fig. 6A). However, analysis of T cell proliferation demonstrated that anergy in T cells induced by M{phi} could be completely reversed by addition of exogenous IL-2 (Fig. 6B). There was a very strong proliferative T cell response in secondary cocultures of T cells with DC of donor A reflecting proliferation levels of a secondary T cell response. In addition, increased proliferation of T cells could be observed in secondary cocultures of T cells with DC of donor B after IL-2 treatment. These results demonstrated that M{phi}-induced anergy in T cells could be completely reversed by addition of exogenous IL-2.


Figure 6
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  		FIGURE 6. T cells were harvested from primary cocultures with allogeneic M{phi} of donor A at day 6 and cultured without (A) or with (B) low dose IL-2. After 5 days, T cells were restimulated in a secondary MLR with different numbers of mDC of donor A (•) or mDC of an unrelated donor B ({circ}). Proliferation of T cells was determined at day 3 by [methyl-3H]thymidine incorporation. The results are representative of five independent experiments.

 
M{phi} induced anergy in resting but not preactivated T cells

To analyze whether M{phi} might also be able to induce anergy in activated T cells, cocultures of T cells and allogeneic mDC were established. After 6 days, T cells were rescued, and a secondary coculture was initiated with M{phi} generated from monocytes of the same donor. After 3 days, T cells were rescued again, and a third coculture was established with mDC. Proliferation of T cells was analyzed in aliquots obtained from each coculture by [methyl-3H]thymidine incorporation. Strong T cell proliferation could be detected in the first coculture with allogeneic DC, demonstrating that T cells were fully activated. Analysis of thymidine incorporation revealed a weak proliferative response in T cells 3 days after initiation of the secondary coculture with M{phi}. In contrast with cocultures with resting T cells, M{phi} did not induce anergy in activated T cells as a high proliferation rate could be observed in T cells at the end of the third coculture with mDC, suggesting that previous activation was sufficient to overcome suppression mediated by macrophages (Fig. 7).


Figure 7
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  		FIGURE 7. Cocultures of T cells and allogeneic mDC were established. After 6 days, T cells were rescued, and a secondary coculture was initiated with M{phi} generated from monocytes of the same donor. After 3 days, T cells were rescued again, and a third coculture was established with mDC. Proliferation of T cells was analyzed in aliquots obtained from each coculture by thymidine incorporation.

 
M{phi} induced T cells with regulatory properties

The finding that coculture of M{phi} with allogeneic T cells resulted in profoundly hyporesponsive T cells suggested that these cells might also have acquired suppressive capacity. We therefore tested the ability of T cells obtained from cocultures with M{phi} to suppress the response of autologous resting T cells upon challenge with allogeneic mDC. Different numbers of mDC were cocultured with 50,000 resting allogeneic T cells alone or in the presence of 50,000 T cells rescued from cocultures with allogeneic M{phi} at day 6 (ratio 1:1), and proliferation was analyzed 6 days after initiation of the cocultures (Fig. 8A). There was an inhibition of >50% in the proliferative response of T cells stimulated with allogeneic DC in the presence of T cells rescued from cocultures with M{phi}.


Figure 8
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  		FIGURE 8. A, Cocultures of 50,000 resting T cells were established with different numbers of allogeneic mDC alone or in the presence of 50,000 autologous T cells rescued from cocultures with allogeneic M{phi} at day 6. Proliferation of T cells was analyzed after 6 days using [methyl-3H]thymidine incorporation. B, To demonstrate cell number-dependent suppression, 10,000 mDC were cocultured with 50,000 resting allogeneic T cells in the presence of different numbers of T cells (10,000–200,000), which had been rescued from cocultures with M{phi}. Proliferation of T cells was analyzed after 6 days using [methyl-3H]thymidine incorporation.

 
To demonstrate a cell number-dependent suppression, 10,000 mDC were cocultured with 50,000 resting allogeneic T cells in the presence of different numbers of autologous T cells, which had been rescued from cocultures with M{phi} (Fig. 8B). Proliferation of T cells was inhibited by T cells rescued from cocultures with M{phi} in a clearly cell number-dependent manner, demonstrating that M{phi} induced T cells with regulatory properties that may contribute to the suppression of T cells mediated by M{phi}.

For further characterization of the regulatory T cell population induced by M{phi}, the phenotypes of CD4+ and CD8+ T cells were determined by FACS-analysis (Fig. 9). Immunophenotyping 6 days after initiation of cocultures with M{phi} revealed that the majority of CD4+ and CD8+ T cells maintained a naive phenotype reflected by expression of CD45RA at high levels and expression of CD45RO in only a minor fraction of CD4+ T cells as observed in untreated control cells. In contrast, CD4+ and CD8+ T cells obtained from cocultures with mDC at day 6 were generally CD45RO+ with only few CD45RA+ cells remaining. No major differences could be observed in the levels of CTLA-4, CD69, or CCR7 expression between CD4+ and CD8+ T cells stimulated with mDC or M{phi}. However, expression of CD25 could be detected in only a significant fraction of CD4+ and CD8+ T cells cocultured with mDC, whereas CD4+ and CD8+ T cells stimulated with M{phi} as well as untreated control cells were generally CD25–. These results indicated that the previously described population of CD4+CD25+ T regulatory cells was not involved in the induction of T cell anergy mediated by M{phi}.


Figure 9
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  		FIGURE 9. Cocultures of T cells were established with allogeneic mDC or M{phi}, and the phenotype of CD4+ and CD8+ T cells as well as resting autologous T cells was determined at day 6 (d6) by FACS using specific mAbs. T0, Time zero; open profiles, isotype control; closed profiles, Ag.

 
To analyze whether IL-10-producing CD4+ T cells were present in cocultures with M{phi}, CD4+ and CD8+ subpopulations were separated at day 6 of coculture, and expression of IL-10 mRNA was determined by RT-PCR. There was a strong expression of IL-10 mRNA in CD4+ T cells obtained from cocultures with M{phi}, suggesting that M{phi} drive the differentiation of IL-10-producing CD4+ type 1 T regulatory cells (Fig. 10).


Figure 10
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  		FIGURE 10. CD4+ and CD8+ T cells were enriched after 6 days of coculture with M{phi} or mDC, and total mRNA was isolated. Expression of IL-10 mRNA and 18S mRNA was analyzed by RT-PCR.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Peripheral T cell tolerance can be induced and maintained by a variety of mechanisms including deletion, induction of T cell hyporesponsiveness, or differentiation and activation of T regulatory cells (22). The present results demonstrate that resting M{phi} are able to suppress allogeneic T cells very efficiently. In addition, T cells rescued from cocultures with M{phi} could not respond to maximal stimulatory signals provided by mDC in secondary cocultures, supporting the conclusion that M{phi} induced anergy in T cells.

Induction of anergy has been observed to occur in T cells after activation of the TCR in the absence of sufficient costimulation (23, 24, 25). However, we propose that induction of anergy in T cells by M{phi} is an active process requiring negative regulatory signals given that our results demonstrate that allogeneic T cells did not proliferate in cocultures with M{phi} even in the presence of anti-CD28 and exogenous IL-2. In contrast, hyporesponsiveness of allogeneic T cells could be reversed by exogenous IL-2. IL-2 treatment resulted in strong T cell proliferation upon restimulation with mDC resembling a secondary T cell response, which is consistent with previous reports demonstrating that IL-2 reverts the anergic state in T cells (26, 27). In addition, high levels of IL-2 produced by T cells during the primary stimulation with allogeneic mDC might overcome the suppressive capacity of M{phi}, which was revealed in repetitive stimulation experiments, demonstrating a strong proliferative response upon restimulation of preactivated T cells rescued from M{phi} with mDC in a third coculture.

Restimulation experiments using third-party DC suggested that T cell anergy was not alloantigen specific. However, additional experiments are required to formally demonstrate that M{phi} induce anergy indeed in Ag-independent manner. If this observation can be confirmed, a general role of resting M{phi} as negative regulators of specific immune responses might be proposed. This hypothesis is supported by reports of other investigators demonstrating that M{phi} of the intestinal mucosa retain avid scavenger and host defense functions but acquire profound inflammatory anergy, thereby promoting the absence of inflammation characteristic of normal intestinal mucosa despite the close proximity of immunostimulatory bacteria (14, 28).

It has been demonstrated that immature DC produce IL-10 in an autocrine fashion and drive the differentiation of IL-10-producing CD4+ type 1 T regulatory cells (4, 29, 30). Analysis of the cytokines produced during the cocultures of M{phi} and allogeneic T cells revealed considerable levels of IL-10. With regard to these observations, additional experiments were performed to evaluate whether the hyporesponsivness of allogeneic T cells mediated by M{phi} was dependent on the induction of regulatory T cells. Cocultures of mDC together with allogeneic T cells and T cells rescued from cocultures with M{phi} demonstrated that T cells derived from cocultures with M{phi} strongly suppressed proliferation of autologous resting T cells. Therefore, M{phi} indeed supported the differentiation of T cells with regulatory properties. Induction of regulatory T cells did not require repetitive stimulation by M{phi} as a population of functional active regulatory T cells already differentiated during the first coculture with M{phi}. These results are in contrast to previous reports demonstrating that induction of regulatory T cells by immature DC was dependent on multiple rounds of coculture (29, 31). Therefore, compared with immature DC, M{phi} possess a strong capacity to induce regulatory T cells, which at least in part might contribute to anergy induction observed in allogeneic T cells.

T cells used in these experiments were not separated into subgroups before establishment of cocultures to reflect physiological conditions most closely. Therefore, the major types of T cells present in the peripheral blood were also present in the cocultures. However, to further elucidate the phenotype of regulatory T cells and the mechanism by which M{phi} induce anergy, immunophenotyping experiments were performed via FACS. These results suggest that the regulatory T cell population differentiating during cocultures with M{phi} were of the CD4+ type 1 T regulatory phenotype secreting IL-10 and not related to CD4+CD25+ regulatory T cells naturally circulating in the peripheral blood as a subfraction of CD4+ T cells. These results are supported by previous reports demonstrating the differentiation of IL-10-producing CD4+ type 1 T regulatory cells in the presence of immature DC (4, 29, 30).

Considering the large numbers of M{phi} present in the different tissues and the results presented in this report, we propose an important role of resting M{phi} to maintain peripheral tolerance by induction of anergy in T cells. In contrast to activated M{phi}, which stimulate T cell Ag specifically, this process is not dependent on signals delivered via the TCR to the T cell and therefore presentation of allo- or autoantigens by M{phi} is not required. With regard to these observations, it is intriguing to speculate that M{phi} contained abundantly in liver tissue might be involved in immunosuppression observed after liver allotransplantation, resulting in the requirement of only minimal or no immunosuppressive therapy in the organ recipients (32).

In conclusion, our data demonstrate that resting M{phi} mediate anergy in T cells, which is at least in part dependent on the induction of T regulatory cells supporting the concept that M{phi} maintain peripheral tolerance of T cells in vivo.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work is supported by Wilhelm Sander-Stiftung Grant 2003.077.1. Back

2 Address correspondence and reprint requests to Dr. Martin Fleck, Department of Internal Medicine I, University of Regensburg, 93042 Regensburg, Germany. E-mail address: martin.fleck{at}klinik.uni-r.de Back

3 Abbreviations used in this paper: DC, dendritic cell; mDC, mature DC; M{phi}, macrophages; MNC, mononuclear cell. Back

Received for publication July 13, 2005. Accepted for publication May 19, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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