Interaction of Mouse Dendritic Cells and Malaria-Infected Erythrocytes: Uptake, Maturation, and Antigen Presentation

Mice, parasite, and pRBC purification

Female C57BL/6 (B6) mice (Charles River Laboratories), ages 8–12 wk, were maintained in the animal facility of the Montreal General Hospital Research Institute in accordance with the guidelines and policies of the Canadian Council on Animal Care. P. chabaudi AS was maintained as previously described (15), and infections were initiated by i.p. injection of 10⁶ P. chabaudi AS pRBCs. To purify pRBCs, heparinized blood was obtained via cardiac puncture from P. chabaudi-infected B6 mice with 30–50% parasitemia and washed twice with PBS. Blood was diluted with PBS (1–2 ml), loaded onto a 74% Percoll (Sigma-Aldrich) density gradient, and centrifuged at 5000 × g for 20 min at room temperature. The top band, containing >96% pRBCs as determined by staining with Diff-Quik (American Scientific Products), was collected. For nRBC controls, heparinized blood from naive mice was loaded onto a 90% Percoll gradient, centrifuged at 5000 × g for 20 min at room temperature, and the top band was collected. Collected cells were washed twice with PBS and resuspended in cell culture medium.

Generation of DCs from BM precursors

DCs were cultured from BM precursors of naive B6 mice using a method adapted from Inaba et al. (16) and Lutz et al. (17) in the presence of GM-CSF supplied by supernatant from a hypoxanthine, aminopterin, and thymidine medium-sensitive Ag8653 myeloma cell line transfected with murine GM-CSF cDNA, which was generated by B. Stockinger (National Institute for Medical Research, Mill Hill, U.K.) (18) and generously provided by G. Carayanniotis (Memorial University of Newfoundland, St. John’s, Canada). Ag8653 cells were cultured under strict G418 selection in RPMI 1640 medium (Invitrogen Life Technologies) with 5% FCS (HyClone), 2 mM l-glutamine (Invitrogen Life Technologies), 0.5 μM 2-ME (Sigma-Aldrich), and 20 μg/ml gentamicin (Sabex). BM cells were flushed from ethanol-sterilized femurs and tibias of mice, passed through a sterile fine wire mesh to remove debris, and washed twice with medium. Red cells were lysed with NH₄Cl lysing buffer. Cells were plated in 6-well plates at a concentration of 2.5 × 10⁵ cells/ml in complete medium consisting of RPMI 1640 medium (Invitrogen Life Technologies) supplemented with 5% FCS (HyClone), 10 mM HEPES (Invitrogen Life Technologies), 20 μg/ml gentamicin (Sabex), 2 mM l-glutamine (Invitrogen Life Technologies), and 0.5 μM 2-ME (Sigma-Aldrich). For the first 3 days of culture, complete medium was also supplemented with 20% Ag8653 culture supernatant. On days 3 and 5 of culture, the plates were swirled gently and 4 ml of supernatant, containing nonadherent cells, was discarded from each well and replaced with 5 ml of fresh complete medium with 10% Ag8653 culture supernatant. To enrich for growing DCs, the top 4 ml of supernatant from each well was discarded on day 7 of culture, and the cell clusters were collected for subculture. Cell aggregates were gently dislodged from the plate by repeated pipetting with PBS and 1% FCS (HyClone). Cells were resuspended at 1.5 × 10⁶ cells/ml in complete medium supplemented with 10% Ag8653 culture supernatant and cultured for an additional 20–24 h at 37°C. Resulting cells were routinely 84–88% positive for CD11c as determined by flow cytometry.

Purification of CD11c⁺ DCs and CD4⁺ T cells from spleen

Spleens from B6 mice were removed aseptically, perfused with PBS and 1% FCS (HyClone), teased gently apart, and pressed through a sterile fine wire mesh. To obtain DCs, cells were suspended in PBS and 5 mM EDTA (Sigma-Aldrich) and separated by loading onto Nycoprep (Axis-Shield) density gradient and centrifuging at 600 × g for 20 min at 4°C. Low-density cells at the interphase were collected, washed twice with PBS, and further purified by positive selection using anti-CD11c microbeads (Miltenyi Biotec). The resulting DCs were routinely ≥85–90% positive for CD11c as determined by flow cytometry. Spleen cells were enriched for CD4⁺ T cells using anti-CD4 microbeads (Miltenyi Biotec) and were ≥85–90% positive for CD4 as determined by flow cytometry.

DC uptake assay and flow cytometric analysis

To stain pRBCs or nRBCs, cells (10⁷ cells/ml) were incubated with 2 μM CFSE (Molecular Probes) in complete medium for 15 min at 37°C. CFSE staining was stopped by adding excess complete medium and washing cells twice with complete medium. BM-derived or splenic CD11c⁺ DCs (10⁶ cells/well) were seeded with CFSE-labeled pRBCs or nRBCs in a 1:20 ratio at a final volume of 200 μl for 4 h at 37°C unless otherwise indicated. DCs were pretreated with cytochalasin D (Sigma-Aldrich) or colchicine (Sigma-Aldrich) dissolved in DMSO (Fischer Scientific) at the indicated concentrations or with 0.2% DMSO control at 37°C for 30 min before the addition of pRBCs or nRBCs. Treatment with cytochalasin D or colchicine did not decrease DC viability as determined by trypan blue exclusion and 7-aminoactinomycin D (BD Biosciences) staining (data not shown). To determine the effect of cytokines on uptake by DCs, splenic CD11c⁺ DCs isolated from naive mice were treated with mouse rIFN-γ (25 ng/ml; R&D Systems) or rIL-10 (200 U/ml; BD Biosciences) in complete medium for 12 h before the addition of pRBCs or nRBCs. Following coculture, noningested red cells were removed by lysis with NH₄Cl lysing buffer, and the remaining DCs were washed twice with PBS, FcR blocked, and then stained with PE-labeled anti-CD11c mAb (clone HL3; BD Biosciences) in sorting buffer consisting of PBS with 1% FCS (HyClone) and 0.05% sodium azide (Sigma-Aldrich). To control for transfer of CFSE from lysed RBCs to DCs during the staining procedure, DCs were added to pRBC or nRBC lysate and analyzed for CFSE expression. The uptake of CFSE-labeled pRBCs or nRBCs was determined by gating cells on SSC vs FL2 for the CD11c⁺ population and analyzing CFSE staining on FL1 using FACSCalibur equipped with CellQuest software (BD Biosciences). Uptake of pRBC by splenic DC subsets was determined by staining CD11c⁺ cells cocultured with pRBCs with PE-labeled mAb to B220 (clone R43-6B2), CD4 (clone GK1.5), and CD8 (clone 53.6.7) and analyzing CFSE expression by these CD11c⁺ subsets.

Light and confocal fluorescence microscopy

BM-derived DCs cocultured with CFSE-labeled pRBCs were analyzed by light and confocal microscopy. For light microscopy, cells were centrifuged at 600 rpm onto glass slides, fixed by 100% methanol, stained with Diff-Quik, dried, and photographed at ×100 magnification using a Zeiss Axioskop 2 plus light microscope (Carl Zeiss). Images were acquired with Bioquant Nova prime software (version 6.710.10MT). For confocal fluorescence microscopy, cells were stained with PE-labeled anti-CD11c Ab and a small aliquot of cells at 10⁶ cells/ml were mounted onto glass slides with fluorescent mounting medium (DakoCytomation), allowed to dry overnight, and observed using a Zeiss Axioskop 2 plus fluorescent microscope. Confocal microscopy images were acquired and analyzed using Q capture software (version 1.60).

Splenic DC maturation, cytokine production, and Ag presentation to T cells

Following overnight coculture of splenic CD11c⁺ DCs with pRBCs or nRBCs, noningested red cells were removed by lysis with NH₄Cl lysing buffer, and DCs were FcR blocked and then stained with PE-labeled mAb (BD Biosciences) to CD11c, I-A^b (clone AF6-120.1), CD40 (clone 2/23), CD80 (clone 16-1041), and CD86 (clone GL-1) in sorting buffer. Cells were gated on the CD11c⁺ and CFSE⁺ population, and the percentages and mean fluorescence intensity (MFI) of cells expressing MHC class II and costimulatory molecules were determined by flow cytometry. To determine DC expression of maturation markers ex vivo before and after P. chabaudi infection, splenic CD11c⁺ DCs purified from naive and day 5-infected mice were stained with mAb as described above and analyzed by flow cytometry. Supernatants of splenic CD11c⁺ DCs (10⁶ cells/well) incubated with pRBCs or nRBCs in a 1:10 ratio for 48 h at 37°C were analyzed for cytokine production by ELISA as described previously (5, 19). To determine Ag presentation function of malaria-pulsed DCs, splenic CD11c⁺ DCs were pulsed with pRBCs or nRBCs at the ratios indicated or incubated in complete medium only as nonpulsed controls. Following overnight culture, noningested RBCs were removed by lysis with NH₄Cl lysing buffer, DCs were washed three times with complete medium, and 100 μl of DCs at 5 × 10⁶ cells/ml was plated with splenic CD4⁺ T cells, freshly purified from naive B6 mice, in varying ratios as indicated at a final volume of 200 μl in 96-well plates for 48 h at 37°C. Proliferation was determined by incorporation of [³H]thymidine during the last 6 h of incubation. Supernatants from DC and T cell cocultures were analyzed for IFN-γ production by ELISA as described previously (19). All pRBC and nRBC preparations used in this study tested negative for the presence of endotoxin using a Limulus amebocyte lysate gel clot test (Sigma-Aldrich) with a sensitivity limit of 0.015 EU/ml. In addition, to control for possible endotoxin contamination, pRBCs and nRBCs were treated with polymixin B (10 μg/ml; Sigma-Aldrich) before incubation with DCs. Results obtained with polymixin-treated pRBCs or nRBCs were identical to those obtained with untreated red cells (data not shown), confirming that the pRBC-induced DC responses observed in this study were not attributable to endotoxin contamination.

Statistical analyses

Data are expressed as mean ± SEM. Statistical significance of differences between experimental groups as indicated was analyzed by two-tailed, unpaired Student’s t test. Statistical significance was defined as follows: ∗, p < 0.05; ∗∗, p < 0.01; and ∗∗∗, p < 0.001. All statistical analyses were performed using SAS/STAT software (SAS Institute).

Preferential uptake of malaria-infected RBCs by BM-derived DCs

To better understand the early interactions between DCs and P. chabaudi-infected RBCs, we developed a flow cytometry technique to measure the uptake of pRBCs or nRBCs by DCs. The intraerythrocytic location of the P. chabaudi AS parasite enabled us to use the dye CFSE to label highly purified pRBCs, as well as nRBCs, and then detect CFSE fluorescence expressed by DCs following coculture. BM-derived DCs were incubated with CFSE-labeled pRBCs or nRBCs, and the percentages of CD11c⁺ DCs expressing CFSE, representing the population of DCs that had ingested the RBCs, were analyzed by flow cytometry. BM-derived DCs were able to ingest pRBCs to a considerably greater extent (>4-fold) than nRBCs (Fig. 1⇓, A and B). The possibility that CFSE inadvertently transferred from lysed RBCs to DCs during the RBC lysis and cell surface staining procedures was observed to be minimal (<1% for nRBCs and 5% for pRBCs; Fig. 1⇓A, right panels). Lysis controls were routinely performed in all experiments, and the values obtained were subtracted from the percentages of uptake presented here. The preferential uptake of pRBCs by DCs was observed as early as 2 h of coculture and peaked at 6 h with no notable increases thereafter at 20 h (Fig. 1⇓C). Even after 20 h of coculture, the percentage of DCs that ingested nRBCs was significantly lower than the percentage that ingested pRBCs.

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FIGURE 1.

DCs preferentially take up pRBCs vs nRBCs. BM-derived DCs were incubated with CFSE-labeled pRBCs or nRBCs in a ratio of 1:20 for 4 h at 37°C. Following coculture, noningested RBCs were lysed, DCs were stained with PE-labeled anti-CD11c mAb, and CFSE fluorescence was determined by flow cytometry. A, Percentages of cells expressing both PE-CD11c (FL2) and CFSE (FL1) following coculture of DCs with intact pRBCs or nRBCs (left panels) or lysis controls (right panels) in which DCs were cocultured with pRBC or nRBC lysate. B, Histograms show CFSE expression by CD11c⁺ DC control (dotted line) or by DCs cocultured with nRBCs (gray line) or with pRBCs (black line). C, Percentages of gated CD11c⁺ cells expressing CFSE after incubation with pRBCs or nRBCs for the time intervals as indicated. Data in A and B are representative of six to eight independent experiments. Data in C are expressed as mean ± SEM pooled from three independent experiments. Statistical analyses compared pRBCs vs nRBCs at each time point (∗∗∗, p < 0.001).

DCs internalize malaria-infected RBCs via actin-dependent phagocytosis

Phagocytosis of large particles (usually larger than 0.5 μm in diameter) by mammalian cells, including macrophages and DCs, requires actin polymerization at the site of entry and subsequent growth of new actin filaments that facilitate cell membrane motility (20). To determine whether the uptake of pRBCs by DCs was mediated by actin-dependent phagocytosis, BM-derived DCs were treated with cytochalasin D, a drug that inhibits actin polymerization and addition of actin monomers to the ends of growing filaments (21), before the addition of pRBCs or nRBCs. As shown in Fig. 2⇓A, DCs treated with cytochalasin D had significantly lower uptake of both pRBCs (54 vs 13%) and nRBCs (7.5 vs 4%) than untreated DCs. This inhibition of uptake was observed at all ratios of DC cocultured with RBCs (Fig. 2⇓B), as well as at all doses of cytochalasin D tested (Fig. 2⇓C). There was no dose response using higher concentrations of cytochalasin D, and the observed reductions were independent of the DMSO used as a diluent, which resulted in similar levels of uptake of pRBCs and nRBCs as untreated DCs (Fig. 2⇓C). Because treatment with cytochalasin D did not completely abrogate the uptake by DCs, some pRBCs may have been phagocytosed via nonactin-dependent mechanisms. Treatment with colchicine, a microtubule depolymerizing agent, did not impair uptake of pRBCs or nRBCs at various concentrations tested (Fig. 2⇓D), suggesting that functional microtubules do not play a major role in the uptake of pRBCs by DCs.

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FIGURE 2.

Treatment with cytochalasin D, but not colchicine, inhibits the uptake of pRBCs and nRBCs by BM-derived DCs. A, Percentages of gated CD11c⁺ cells expressing CFSE following no treatment (left panels) or treatment with cytochalasin D (right panels). Histograms show CFSE expression by CD11c⁺ DCs incubated without RBCs (line) or with pRBCs or nRBCs (shaded area). B, Treatment with cytochalasin D (CyD; 8 μM) inhibits uptake of pRBCs and nRBCs at all ratios of DCs incubated with RBCs. C, Cytochalasin D inhibits the uptake of pRBCs and nRBCs at all concentrations tested. Percentages of inhibition relative to no treatment (0 μM) are indicated. D, Treatment with colchicine does not affect uptake of pRBCs or nRBCs at any concentration tested. Data are expressed as mean ± SEM pooled from four (B and C) or two (D) independent experiments. Statistical analyses compared uptake of pRBCs with no treatment vs treatment with cytochalasin D at each DC:RBC ratio (∗∗, p < 0.01; ∗∗∗, p < 0.001).

To demonstrate internalization of pRBCs by DCs, cytospins of Diff-Quik-stained BM-derived DCs cocultured with pRBCs were examined by light microscopy. Following 2–6 h of coculture and lysis of noningested red cells, intact pRBCs were observed inside cytoplasmic compartments of BM-derived DCs that resemble phagosomal vacuoles (Fig. 3⇓A). To confirm that the expression of CFSE fluorescence by gated CD11c⁺ DCs truly represents internalization of pRBCs by DCs rather than only attachment of CFSE-labeled cells to the CD11c⁺ cell surface, we examined colocalization of CFSE-labeled pRBCs and PE-stained CD11c⁺ cells by confocal microscopy (Fig. 3⇓, B–D). In representative confocal fluorescent microscopy images, CFSE-labeled pRBCs are shown to colocalize with three PE-labeled CD11c⁺ DCs (Fig. 3⇓D). Collectively, these microscopy images provide strong evidence that flow cytometric analyses of CFSE expression by gated CD11c⁺ DCs using the assay we developed were an accurate and objective measurement of internalization by DCs of CFSE-labeled pRBCs or nRBCs.

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FIGURE 3.

Internalization of pRBCs by BM-derived DCs and colocalization of DCs and CFSE-labeled pRBCs. A, Diff-Quik-stained light microscopic images show pRBCs, indicated by arrows, internalized by DCs. B, Red fluorescence of PE-labeled CD11c⁺ DCs. C, Green fluorescence of CFSE-labeled cells, representing CFSE-labeled pRBCs inside DCs. D, Merged confocal microscopic image showing colocalization of CFSE-labeled pRBCs and CD11c⁺ DCs. Light microscopic images and confocal fluorescent images were acquired at ×100 and ×40 magnification, respectively. Data shown are representative of two independent experiments.

Uptake of malaria-infected RBC by splenic CD11c⁺ DCs is selectively enhanced following malaria infection in vivo

Next, we used our flow cytometric technique to study the uptake of pRBCs by mouse spleen DCs before and during infection with P. chabaudi AS. Although we used BM-derived DCs to standardize our flow cytometric uptake assay and showed that these DCs exhibited preferential uptake of pRBCs, it is the DC population in the spleen that is of major interest because of the central role of this organ in filtering pRBCs from the blood and in generating a protective immune response to blood-stage malaria (22). As observed with BM-derived DCs, splenic CD11c⁺ DCs showed significantly higher (>4-fold) uptake of pRBCs than nRBCs (Fig. 4⇓A) at all time points before and after P. chabaudi infection (Fig. 4⇓B). Importantly, the percentage of splenic CD11c⁺ DCs ingesting pRBCs increased following P. chabaudi infection (47.7% at day 5 postinfection (p.i.) vs 18.6% at day 0; Fig. 4⇓A). The uptake of pRBCs by splenic CD11c⁺ DCs increased markedly at days 2–5 p.i. and declined thereafter to preinfection levels by day 8 p.i. (Fig. 4⇓B), indicating that maximal uptake of pRBC ex vivo by splenic DCs preceded peak parasitemia in P. chabaudi-infected mice. In contrast, the uptake of nRBCs increased only slightly after infection (5% higher at days 5 vs 0 p.i.) and was significantly lower than the uptake of pRBCs at all time points before and after infection. Uptake of pRBCs and nRBCs by splenic DCs from naive mice was significantly impaired by treatment with cytochalasin D at all ratios of DCs incubated with pRBCs or nRBCs (55–65% inhibition; Fig. 4⇓C) but was not affected by treatment with colchicine (data not shown). Therefore, splenic DCs selectively recognize and phagocytose pRBCs, and this preferential uptake of pRBCs is enhanced in the early days of blood-stage malaria infection in vivo. These results, while specific to interactions of DCs with malaria-infected RBCs, are consistent with the sentinel role of DCs in detecting invading pathogens and capturing foreign Ags during acute infection.

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FIGURE 4.

Splenic CD11c⁺ DCs preferentially take up pRBCs vs nRBCs at increasing levels in the early days following P. chabaudi infection in vivo. A, Splenic DCs enriched from naive (left panels) or day 5-infected (right panels) B6 mice were incubated with CFSE-labeled pRBCs or nRBCs, and the level of uptake by nongated cells (dot plots) or gated CD11c⁺ cells (histograms) was determined by CFSE fluorescence as detected by flow cytometry. B, Kinetics of pRBC or nRBC uptake by splenic DCs enriched from mice before and after P. chabaudi infection and course of parasitemia in mice infected with P. chabaudi during the same experimental period. C, Splenic DCs preferentially take up pRBCs vs nRBCs at all ratios of DCs incubated with RBCs and uptake is inhibited by treatment with cytochalasin D (CyD, dotted lines). D, Uptake of pRBCs is mediated by CD4⁺, CD8⁺, and B220⁺ subsets of splenic CD11c⁺ DCs before and after infection. E, Pretreatment of splenic DCs from naive or day 5-infected mice with rIFN-γ or rIL-10 in vitro does not affect the uptake of pRBCs or nRBCs compared with untreated DCs. Data in B–D were pooled from three independent experiments each containing three to four mice per group and are presented as mean ± SEM. Data in E are representative of two independent experiments each containing three to four mice per group. Statistical analyses in B and C compared pRBCs vs nRBCs at each time point or DC:RBC ratio and in D compared days 0 vs 5 p.i. for each subset (∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001).

Given that the mouse CD8⁺ DC subset has been shown to selectively endocytose apoptotic cells (14), we questioned whether the level of pRBC uptake differed among the CD4⁺, CD8⁺, and B220⁺ (plasmacytoid) subsets of splenic CD11c⁺ DCs. Following coculture of pRBCs with splenic CD11c⁺ DCs purified from naive or infected mice, DCs were stained with fluorochrome-labeled Abs to CD4, CD8, or B220, and the percentage of CD11c⁺ cells expressing CFSE fluorescence was determined for each DC subset by flow cytometry. All three subsets exhibited phagocytosis of pRBCs before and after acute P. chabaudi infection (Fig. 4⇑D). The CD4⁺ and CD8⁺ DC subsets from infected mice showed significantly higher uptake of pRBCs than those from naive mice, while uptake by the B220⁺ subset was significantly lower after infection.

Effect of cytokines on uptake of malaria-infected RBCs by splenic CD11c⁺ DCs

Previous work in our laboratory showed that rIFN-γ activates macrophages and increases their phagocytosis of pRBCs while rIL-10 has the opposite effect (11). We questioned whether phagocytosis of pRBCs by DCs is similarly controlled by cytokines with opposing immunoregulatory effects. Splenic CD11c⁺ DCs from naive or day 5-infected mice were treated with rIFN-γ, rIL-10, or not treated (medium) for 12 h before the addition of pRBCs or nRBCs as described previously (11). As shown above, splenic DCs from infected mice demonstrated higher levels of pRBC uptake than DCs from naive mice (Fig. 4⇑E). However, treatment with rIFN-γ or rIL-10 did not significantly affect the uptake of pRBCs or nRBCs by DCs from either naive or infected mice, suggesting that uptake of pRBCs by DCs is not dependent on the cytokines present in the spleen microenvironment.

Uptake of malaria-infected RBC by splenic CD11c⁺ DCs induces maturation, cytokine production, and CD4⁺ T cell activation

The aforementioned flow cytometric analyses were conducted on total CD11c⁺ DC populations cocultured with pRBCs and included both phagocytic and nonphagocytic DCs. We then determined whether preferential uptake of pRBCs by DCs induces maturation and presentation of Ags to CD4⁺ T cells. Following an overnight DC uptake assay, splenic CD11c⁺ DCs that had captured pRBCs showed significantly higher expression of MHC class II and costimulatory molecules than DCs that had ingested nRBCs as demonstrated by the higher percentages of cells expressing these molecules (Fig. 5⇓A) and by the higher levels of MFI (Fig. 5⇓B). The ability of pRBCs, but not nRBCs, to stimulate maturation of splenic DCs in vitro was consistent with the observation of in vivo maturation after P. chabaudi infection (Fig. 5⇓C). Because uptake of pRBCs by DCs was associated with phenotypic maturation, we assessed whether DCs that had phagocytosed pRBCs compared with DCs that had ingested nRBCs were also functionally more mature in terms of cytokine production and stimulation of CD4⁺ T cell responses. Splenic DCs cocultured with nRBCs or in medium alone did not produce detectable levels of IL-12p40 or, importantly, bioactive IL-12p70 compared with DCs pulsed with pRBCs (Fig. 6⇓, A and B). In addition, splenic DCs pulsed with pRBCs produced significantly more IL-2 (Fig. 6⇓C) and stimulated significantly higher levels of CD4⁺ T cell proliferation (Fig. 6⇓E) and IFN-γ production (Fig. 6⇓F) than did splenic DCs pulsed in medium alone (nonpulsed) or with nRBCs. Notably, stimulation of T cell proliferation and IFN-γ production increased in a dose-dependent manner when DCs were pulsed with a higher number of pRBCs (Fig. 6⇓, E and F). The lower stimulation of T cell responses by DCs pulsed in medium alone or with nRBCs was not associated with the production of anti-inflammatory IL-10, which was significantly lower than the level produced by DCs pulsed with pRBCs (Fig. 6⇓D). Several lines of evidence indicate that pRBC is a poor T cell mitogen in the absence of presentation by an APC such as the splenic DCs used in this study: 1) CD4⁺ T cells incubated with pRBCs alone showed minimal proliferation and failed to produce detectable IFN-γ (Fig. 6⇓, E and F); 2) DCs fixed with 1% paraformaldehyde before pulsing with pRBCs were unable to stimulate CD4⁺ T cells (data not shown); and 3) Th2-type cytokines, IL-4 and IL-13, were not detected in any of the supernatants from DC single cultures or cocultures with T cells (data not shown). These results suggest that the selective interaction of splenic CD11c⁺ DCs with pRBC induced DCs to mature, to produce IL-12, and to promote CD4⁺ T cell proliferation and differentiation into IFN-γ-secreting cells.

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FIGURE 5.

Uptake of pRBCs, but not of nRBCs, by splenic DCs induces maturation. Splenic CD11c⁺ DCs from naive B6 mice were pulsed with CFSE-labeled pRBCs or nRBCs. Following coculture overnight, noningested RBCs were removed, and DCs were stained with PE-labeled Abs to I-A^b (MHC class II), CD40, CD80, and CD86. A, Histograms show PE fluorescence of various markers by gated CFSE⁺ cells. Higher percentages of DCs cocultured with pRBCs expressed MHC class II and costimulatory molecules compared with DCs cocultured with nRBC. B, MFI of the expression of MHC class II and costimulatory molecules by gated CFSE⁺ cells. DCs pulsed with pRBCs expressed higher levels of MHC class II and costimulatory molecules than did DCs pulsed with nRBCs. C, MFI of the expression of maturation markers by splenic CD11c⁺ DCs ex vivo at days 0 and 5 p.i. Data in B and C are presented as mean ± SEM and represent two independent experiments of three to four mice per group. Statistical analyses compared pRBCs vs nRBCs in A and days 0 vs 5 p.i. in B for each marker MFI (∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001).

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FIGURE 6.

Splenic CD11c⁺ DCs exposed to pRBCs but not to nRBCs produce cytokines (A–D) and stimulate CD4⁺ T cell proliferation (E) and IFN-γ production (F). CD11c⁺ DCs were enriched from naive B6 mice and pulsed overnight with pRBCs or nRBCs. Following pulsing and removal of noningested RBCs, DCs were washed and incubated in medium alone, with nRBCs or pRBCs, or with freshly purified splenic CD4⁺ T cells for 48 h at 37°C. DCs pulsed with pRBCs produced significantly higher levels of IL-12p40 (A), bioactive IL-12p70 (B), IL-2 (C), and IL-10 (D) than DCs pulsed in medium alone or with nRBCs as determined by ELISA. E, DCs pulsed with pRBCs at ratios of 1:10 or 1:20 stimulated significantly higher levels of CD4⁺ T cell proliferation than nonpulsed DCs or DCs pulsed with nRBCs. F, DCs pulsed with pRBCs stimulated significantly higher levels of IFN-γ production by CD4⁺ T cells than nonpulsed DCs or DCs pulsed with nRBCs. E and F, CD4⁺ T cells cultured with pRBCs alone (1:10 ratio) were included as controls. Data are presented as mean ± SEM of three to four mice per group and are representative of three independent experiments. Statistical analyses in A–D compared pRBCs vs nRBCs for each cytokine and in E and F compared DCs + pRBCs (1:20) vs DCs + nRBCs (1:20) at each DC:T cell ratio (∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001).