Inhibitory effects of rat bone marrow-derived dendritic cells on naïve and alloantigen-specific CD4+ T cells: a comparison between dendritic cells generated with GM-CSF plus IL-4 and dendritic cells generated with GM-CSF plus IL-10
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Unlike mouse immature bone marrow (BM)-derived dendritic cells (DC), rat immature BMDC have not been thoroughly characterised in vitro for the mechanisms underlying their suppressive effect. To better characterise these mechanisms we therefore analysed the phenotypes and immune inhibitory properties of rat BMDC generated with GM-CSF plus IL-4 (= IL-4 DC) and with GM-CSF plus IL-10 (= IL-10 DC).
Both IL-4 DC and IL-10 DC exhibited lower surface expression of MHC class II and costimulatory molecules than mature splenic DC. They had a strong inhibitory effect on responsive T cells in vitro and despite their weak function as antigen-presenting cells they induced anergic T cells. However, only anergic T cells induced by IL-4 DC had a suppressive effect on responsive T cells. Induction of suppressive/tolerogenic T cells by IL-4 DC required direct contact between antigen-specific T cells and IL-4 DC. In addition, IL-4 DC and IL-10 DC prolonged allograft survival in an antigen-specific manner.
A unique phenotype of immature BMDC was isolated from the cultures. The mechanisms underlying the suppressive effect may be caused by their inability to deliver adequate costimulatory signals for T-cell activation. In addition, IL-4 DC but not IL-10 DC induce anergic T cells with suppressive function. This indicates that IL-4 DC and IL-10 DC may differ in the quality of their costimulation although no differences in the surface expression of costimulatory molecules were found.
KeywordsDendritic Cell Granulocyte Macrophage Colony Stimulate Factor Costimulatory Molecule Allograft Survival Heart Allograft
In recent years it has become clear that dendritic cells (DC) are not only potent inducers of adaptive immune responses, but also essential mediators in the induction and maintenance of T-cell tolerance . The biological properties of DC depend on their phenotypically distinct states of development . Their delaying effect on allograft rejection has been demonstrated in several rodent models [reviewed in ].
Mouse and human DC have both been studied thoroughly [reviewed in ]. Rat DC have been investigated particularly by groups interested in transplantation research [5, 6, 7]. They were not studied thoroughly, although established culture methods exist for the generation of bone marrow-derived rat DC (BMDC) [8, 9]. The maturation of BMDC varies from species to species despite comparable culture conditions. In mice, for example, low doses of granulocyte macrophage colony stimulating factor (GM-CSF) combined with interleukin (IL)-4 induce the formation of mature BMDC , whereas in rats the same combination produces immature BMDC . The effect of GM-CSF and IL-10 on the generation of rat BMDC is not clearly known.
In the present study we examined the ability of IL-4 DC and IL-10 DC to inhibit both the activation of naïve T cells and the restimulation of antigen-specific T cells in vitro. We also analysed their in vivo potential to prolong allograft survival.
Generation of rat BMDC
Femur and tibia bones of young (8–10 weeks) Lewis rats were extracted and disinfected in 70% ethanol. Both ends of the bones were cut and the bone marrow (BM) cells were flushed with 20 ml phosphate-buffered saline (PBS). The BM cells were cultured at a cell density of 5–8 × 105 cells/ml in culture dishes (Falcon, Becton Dickinson Biosciences). The RPMI 1640 culture medium  was supplemented with 5 ng/ml recombinant rat GM-CSF (R&D Systems, Heidelberg, Germany) and 5 ng/ml recombinant rat IL-4 (Miltenyi Biotech GmbH, Germany) or 5 ng/ml rat IL-10 (Miltenyi Biotech GmbH). On day 6, non-adherent cells and cells growing in clusters were collected.
Activation of naïve T cells and restimulation of antigen-specific T cells
Naïve T cells (105 cells/well) were incubated with 20 Gy irradiated IL-4 DC, IL-10 DC, or mature S-DC (104 cells/well) for 3 days at 37°C in a 5% humidified CO2 atmosphere. Allopeptide P1-specific T cells (105 cells/well) were incubated with irradiated (20 Gy) IL-4 DC, IL-10 DC, or S-DC (104 cells/well) loaded with P1 (1.25 μg/well) for 3 days at 37°C in a 5% humidified CO2 atmosphere. T-cell proliferation in 96-well, round-bottom plates was measured after 3H-thymidine (0.5 μCi/well) incubation for the last 6 h before harvesting. Radioactivity was determined as previously described . Results (mean ± SD) were expressed in counts per minutes (cpm).
Some of the assays were performed in 96-well transwell plates (Corning Life Sciences, The Netherlands). The upper compartment contained immature rat BMDC (1 × 104), the lower compartment antigen-specific T cells (1 × 105). After 3 days of culture, the transwells were removed and P1-loaded S-DC (104/well) as well as antigen-specific T cells (1 × 105/well) were added to the lower compartments. The cultures were then incubated for another 3 days and pulsed with 0.5 μCi/well [3H]-thymidine for the last 6 h of culture. The incorporation of [3H]-thymidine was measured as described .
Heterotopic heart transplantation with and without antigen-loaded BMDC
The animal experiments were conducted in accordance with European, national and institutional animal care policies. Ten million BMDC were incubated with 20 μg P1 for 30 min in 500 μl PBS, washed 2 times with PBS and transferred intravenously via the penile vein into Lewis rats under full anesthesia with isoflurane 1 day before transplantation. Fully vascularised, heterotopic heart transplantation was performed, and graft survival was monitored as described .
IL-4 DC and IL-10 DC exhibited an identical phenotype
IL-4 DC and IL-10 DC neither activated naïve T cells nor restimulated antigen-specific T cells
Primers and PCR conditions.
Sequence (5'- > 3')
For: GGT CGG TGT GAA CGG ATT TG
Rev: GTG AGC CCC AGC CTT CTC CAT
For: CAG GAT CTG GAA GGT CCA
Rev: AGC TGT GGT TGT GCT GA
For: CGC TAT GGG GCT GCT TGT TGA CAG;
Rev: GAC GGT ATC AGT GGT CTC AGT GGC
For: TGG TGA AAC ACC TGA CCA
Rev: GTT TCT CTG CTT GCC TCA
For: TGG GAA ACA GAG CTC TCA
Rev: AGG TTG ATC GAC TCG TCA
In the BMDC-uninfluenced T-cell proliferation assay, the strongest increase in proliferation of antigen-specific T cells occurred between days 2 and 3 of the 3-day culture. The addition of P1-pulsed IL-4 DC or IL-10 DC to the cultures on day 2 halted this strong increase in T-cell proliferation within 24 h (Fig. 3E, F).
IL-4 DC-T and IL-10 DC-T showed anergic properties
IL-4 DC-T had an inhibitory effect on antigen-specific T cells
P1-loaded IL-4 DC and IL-10 DC prolonged antigen-specific cardiac allograft survival
The effects of allopeptide pulsed and unpulsed IL-4 DC and IL-10 DC in vivo.
Origin of DC
Donor of heart allografts
Survival time (d) of heart allografts in LEW recipients
MST ± SD (d)1)
1 Syngeneic Ctr
2 Syngeneic Ctr + P1
3 Allogeneic Ctr I
7.3 ± 0.5
4 Allogeneic Ctr I + P13)
5.3 ± 0.5
5 1 × 107 IL-4 DC
6.8 ± 0,5
6 1 × 107 IL-4 DC + P1
9.5 ± 1.0
7 1 × 107 IL-10 DC
7.1 ± 0.4
8 1 × 107 IL-10 DC + P1
8, 9(×2), 10(×5)
9.5 ± 0.8
9 Allogeneic Ctr II
10 Allogeneic Ctr II + P1
7.3 ± 0.5
11 1 × 107 IL-4 DC
12 1 × 107 IL-4 DC + P1
7.3 ± 0.6
13 1 × 107 IL-10 DC
14 1 × 107 IL-10 DC + P1
In the present study, we compared the effects of 2 types of rat BMDC on the proliferation of naïve and antigen-specific T cells in vitro and on the survival of allogeneic heart allografts. Both DC types displayed lower surface expression of MHC class II and costimulatory molecules compared to mature splenic DC. IL-4 DC and IL-10 DC had a strong inhibitory effect on responsive T cells. This suppressive effect was detectable within 24 h after the BMDC were added to cultures with antigen-specific T cells and mature splenic DC (Fig. 3E, F). To our knowledge this is the first description of the time course BMDC needed to suppress T-cell proliferation. We found that antigen-specific T cells became anergic after incubation with IL-4 DC and IL-10 DC. The same effect has been described for human IL-10 modified DC on CD4+ T cells . Anergic T cells isolated from cultures with P1-loaded IL-4 DC suppressed the DC-mediated activation of responsive T cells in a cell count-dependent manner. They share this suppressive effect with regulatory T cells . We hypothesise that IL-4 DC and IL-10 DC may differ in the quality of their costimulation, with IL-4 DC inducing suppressive IL-4 DC-T whereas IL-10 DC do not. It should be emphasised, however, that we found no differences in the surface expression of costimulatory molecules between the two BMDC types.
The results of transwell experiments showed that the contact between IL-4 DC and the antigen-specific T cells is a prerequisite for inducing suppressive IL-4 DC-T. However, we made no attempt in this study to determine whether the anergic IL-4 DC-T mediate their suppressive effect via cell-cell contact or by soluble factors. Vendetti et al., for example, reported that inhibition mediated by anergic murine T cells is dependent on cell-cell contact . They also described an inhibitory effect of anergic T cells on the antigen-presenting function of mature DC.
IL-4 DC and IL-10 DC, loaded with allopeptide P1, prolonged allograft survival (Fig. 6). In addition, survival time can be improved by increasing the number of transferred cells. The application of 30 million P1-pulsed IL-10 DC, for example, prolonged survival time to a median of 10.6 ± 0.8 days (not shown) from the 9.5 ± 0.8 days achieved with 10 million P1-pulsed IL-10 DC. Allograft survival was prolonged only when the BMDC were pulsed with the immunodominant allogeneic peptide P1 involved in allograft rejection . Our results accord with those of Chowdhury et al. , who showed that the presentation of allogeneic peptides by tolerogenic thymic rat DC greatly prolongs allograft survival. Compared to the results of Chowdhury et al., our 2–3 day prolongation of allograft survival may seem meager, but considering the strength of the allogeneic immune response induced by alloreactive T cells and the fact that no immunosuppressive drugs were used, our findings appear very promising.
The data suggest that rat IL-4 DC and IL-10 DC have suppressive/regulatory properties comparable to those described for immature mice BMDC. They demonstrate a strong inhibitory effect on responsive T cells, probably the consequence of a reduced surface expression of costimulatory molecules paired with the inability to deliver adequate costimulatory signals to T cells. IL-4 DC and IL-10 DC are identical in phenotype and in some of their effects, but they are different in their capacity to induce suppressive T cells. IL-4 DC induce T cells with suppressive/regulatory function whereas IL-10 DC do not. This may indicate that IL-4 DC and IL-10 DC differ in the quality of their costimulation. Further studies are necessary to test this hypothesis.
This study was supported in part by funds from the Federal Ministry of Education and Research, granted to the Interdisciplinary Centre for Clinical Research (IZKF) of the University of Würzburg (research project grant number 01 KS 9603) and by the Graduate College 520 (Immunomodulation) of the German Research Foundation (DFG). George Christian Tiurbe, M.D., is a former recipient of a fellowship from the DFG and Anja Matuschek, M.Sc., is presently a recipient of a DFG fellowship.
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