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. Author manuscript; available in PMC: 2011 Aug 15.
Published in final edited form as: J Immunol. 2010 Jul 19;185(4):2140–2146. doi: 10.4049/jimmunol.1000642

Dendritic cells continue to capture and present antigens after maturation in vivo

Scott B Drutman 1, E Sergio Trombetta 1
PMCID: PMC2928255  NIHMSID: NIHMS227429  PMID: 20644175

Abstract

Dendritic cell (DC) maturation is critical for the regulation of T-cell responses. The down regulation of endocytosis upon maturation is considered a key adaptation that dissociates prior antigen capture by DCs from subsequent T-cell engagement. In order to study the dynamics of antigen capture and presentation in situ, we studied the capacity for antigen uptake by DCs matured in their natural tissue environment. We found that after maturation in vivo mouse DCs retained a robust capacity to capture soluble antigens. Furthermore, antigens internalized by mature DC were efficiently presented on MHC-class II and cross-presented on MHC-class I. These results suggest that under inflammatory conditions mature DC may contribute to T-cell stimulation without exclusively relying on prior exposure to antigens as immature DC precursors.

INTRODUCTION

Dendritic Cells (DCs) are critical antigen presenting cells (APC) characterized by their efficient conversion of internalized antigens into peptide-MHC complexes (pMHC) required to orchestrate T-cell responses (1-3). Critical DC functions such as antigen presentation, expression of co-stimulatory molecules and migration are tightly controlled by a maturation process, where stimuli such as Toll-like receptor (TLR)-ligands or inflammatory cytokines convert immature DCs into mature DCs specialized for T-cell stimulation (3, 4).

Manipulation of DC maturation shows high therapeutic potential, especially in vaccination regimes where antigen delivery to DCs is coordinated with a maturation stimulus to optimize T-cell stimulation (1, 5). Due to the paucity of DCs and the consequent difficulty in tracking their APC function in situ, many of the studies of DC maturation have relied on culture systems amenable for DC manipulation and analysis. These studies have shown that DC maturation is accompanied by a marked reorganization of endocytic compartments(6-8) and a concomitant down-regulation of antigen capture (9-19). Such restriction of antigen capture to immature DCs is considered to contribute to the functional distinction between immature and mature DCs, allowing mature DCs to focus on presenting to T-cells antigens acquired before or at the time that DC receive maturation stimuli (20-22) with some reports of receptor-mediated uptake by cultured DCs (23-28). However, despite the important role envisioned for the modulation of antigen capture upon DC maturation, this regulatory step has not been defined in vivo. This lead us to examine the dynamics of antigen capture by DCs during maturation in situ, in their natural tissue environment. We found that the capacity to capture antigens does not appear to be significantly modified when DC mature in vivo. Furthermore, antigens internalized by mature DC can be efficiently processed and presented on both MHC-I and MHC-II.

Materials and Methods

Mice

C57Bl/6 (B6), OT-I/RAG1 (OT-I), OT-II2.a/RAG1 (OT-II), C3H/HeN (C3H), B6.SJL (CD45.1) and Abb (MHC-II-KO) mice (29) mice were from Taconic Farms. B6.CH2bm1/ByJ (BM1) were from The Jackson Laboratory. Mice were housed under specific-pathogen-free conditions and maintained in compliance with institutional and federal regulatory guidelines.

Reagents

PBE is PBS, 0.5% BSA (endotoxin free, Equitech-Bio), 1 mM EDTA, pH 7.4. Complete RPMI is RPMI (Gibco), 10% Heat-inactivated FBS (endotoxin free, Invitrogen), non-essential amino acids, 110 μg/ml Sodium-Pyruvate, 2 mM LGlutamine, 100units/ml Penicillin, 100 μg/ml Streptomycin (Gibco), and 100 μM b-Mercaptoethanol (Sigma). For preparation of bacterial lysate, DH5 K-12 E. coli (Invitrogen) were grown overnight in LB media, washed 3 times in PBS, resuspended at 3×109 bacteria/ml, heat killed at 80°C for 45 min, subjected to 5 freeze/thaw cycles and finally passed though a fine gauge needle 3 times to disrupt clumps. The lysate contained ~6×105 EU/ml (~10μg/ml) endotoxin by LAL test (Cambrex).

Cells

For isolation of DCs, spleens were digested with Liberase Blendzyme 2 (Roche Diagnostics) for 15 min in PBS at 21°C, passed through a 40 μm cell strainer, treated with ACK Buffer (Lonza) to remove red cells, and resuspended in PBE. DCs were first enriched to 30-50% by magnetic negative depletion by incubating splenocytes with biotinylated CD19 (MB19.1), CD3 (145-2C11), NK1.1 (PK136), Ly-6G/Gr-1 (RB6-8C5), and erythroid cell marker (TER-119) antibodies (eBioscience), followed by enrichment using the EasySepTM biotin selection kit (StemCell Technologies Inc.). DCs were subsequently sorted on a Dako MoFlo. Post-sort analysis confirmed purity of 99% and viability of 95%. OT-I or OT-II cells were isolated from the lymph nodes and spleens of OT-I/RAG1 KO or OT-II/RAG1 KO mice by disruption through a 40 μm cell strainer, followed by negative selection using mouse CD8+ T-cell or mouse CD4+ T-cell enrichment kit, respectively (StemCell Technologies Inc.). Enriched T-cells were pulsed with 0.5 mM CFSE (Invitrogen) for 5 min, washed twice and resuspended in PBS for adoptive transfers or complete media for T-cell stimulation assays.

Preparation of GFP-OT

GFP-OT construct (Supplementary Fig. 4) in pET-28 vector (Novagen) was transformed into BL21 E. coli (Novagen). Bactieria were grown in TB media (Invitrogen) at 37°C until they reached ~0.1 Absorbance Units at 600 nm, then at 24°C for 16 h with 1mM IPTG (Sigma), spun down at 8,000×g for 15 min, resuspended in 50mM Tris pH 8.0, 500mM NaCl, 50 μg/ml Lysozyme (Sigma), incubated for 30 min, and PMSF and Benzamidine were added to 10 μg/ml (Sigma). After 3 rounds of −80°C freeze/37°C thaw and sonication, the lysate was centrifuged at 20,000×g for 30 min, and the supernatant 0.22μm filtered. Imidazole was added to 20mM, GFP-OT was affinity purified on Ni-Sepharose (GE Healthcare), and eluted in 50mM Tris pH 8.0, 200mM NaCl, 500mM Imidazole. The eluate was diluted 10 fold with H2O, adjusted to 9.5 pH with NaOH, bound to Q-Sepharose (Pharmacia), washed with 0.5% NP-40, 50 mM Tris pH 9.5 to remove endotoxin, washed with 50 mM Tris pH 9.5 to remove all detergent, eluted with 500 mM NaCl, 50 mM Tris pH 9.5, and dialyzed against PBS. The resulting GFP-OT protein had <1.26 EU/mg (< ~125pg/mg) endotoxin by LAL test (Cambrex).

Maturation of DCs

Maturation protocols were chosen to provide the maximum level of inflammation achievable with these reagents as judged by upregulation of maturation markers on DCs. Sterile, endotoxin-free PBS was used for control injections. For maturation of DCs with TLR agonists, 1μg of LPS (Salmonella enterica serotype typhimurium, Sigma) or 20 nmoles of CpG-B (ODN 1668 5′-tccatgacgttcctgatgct-3′ with phosphorothioate bonds, Invitrogen) were injected intra-peritoneally 16 h prior to experiments. For in vivo bacterial lysate mediated maturation, 200 μl of E. coli lysate (~6x107 bacteria, containing ~2ug LPS) was injected intra-peritoneally 16 h prior to experiments.

Endocytosis assays

For in vitro endocytosis assays, 1×107cells/ml were incubated with GFP-OT (100 μg/ml) at 37°C (or kept on ice for negative controls) for 30 mintues in complete RPMI. All cells were washed 3x in PBE, before analysis by flow cytometry. For in vivo endocytosis assays, mice were injected intraperitoneally with 0.5 to 2mg of GFPOT and 30 minutues later, spleenocytes were collected and analyzed for antigen capture as compared to a similarly treated mouse not injected with GFP-OT.

In vitro antigen presentation assays

To assay presentation of antigen captured by DC in vivo, 0.5-to-2.0mg GFP-OT or ovalbumin was injected intravenously into mice, and 30 min later DCs were purified as described. Various numbers of DCs were co-incubated in U-bottom 96-well plates with 50,000 CFSE labeled OT-I CD8+ T-cells or OT-II CD4+ T-cells in complete RPMI to allow for a variety of T-cell to DC ratios. 60 h later, T-cell proliferation was assessed by dilution of CFSE using flow cytometry. Additionally, supernatants from these incubations were harvested to measure IFN-γ secreted during T-cell stimulation using Bio-Plex Pro cytokine assays (BioRad). Cytolytic activity of OTI T-cells was measured by the ability to lyse target cells in an antigen-specific manner. Target splenocytes isolated from CD45.1 mice were labeled with high (2 μM) or low (0.4 μM) concentrations of CFSE. The CFSElow labeled cells were pulsed with 100 ng/ml of SIINFEKL peptide (antigen-specific targets), while the CFSEHigh cells were pulsed with 100 ng/ml of RGYVYQGL peptide (VSV nucleoprotein, non-specific). Both populations (CFSEhigh/SIINFEKL & CFSElow/VSV) were combined in equal numbers and mixed with the expanded OT-I cells derived from the antigen presentation assays (20:1 effector:target ratio; enumerated by number of cells initially added). After 6 hours of incubation, the mixture of OT-I and CD45.1 targets was analyzed by flow cytometry to quantify the target cells reamining (CD45.1+). % specific lysis was determined as 100 minus the percentage of CFSEhigh/SIINFEKL relative to the CFSElow/VSV cells.

In vivo antigen presentation assays

Mice were injected intra-peritoneally with maturation stimuli or vehicle control, and intravenously with 1×106 CFSE labeled OT-I and/or OT-II cells. 16-40 h later, mice were injected intravenously with 200 μg of ovalbumin. 60 h later, spleenocytes were isolated and T-cell proliferation was analyzed by flow cytometry. In other experiments, 500,000 purified immature or mature DCs (isolated by cell sorting) were adoptively transferred intravenously into BM1 mice along with 1×106 CFSE labeled OT-I T-cells. Alternatively, 500,000 purified immature or mature spleen DCs (isolated by cell sorting as described in Figure S1) were adoptively transferred by intravenous injection into Abb mice (MHC-II KO) along with 1×106 CFSE labeled OT-II T-cells. 1 h later, the mice were injected with 1 mg of OVA. 60 h later, spleenocytes were isolated and T-cells proliferation was analyzed by flow cytometry. To Measure serum anti-OVA IgG, Costar type 2592 plates coated with OVA were incubated with sera, and IgG was detected with HRP-conjugated Goat anti-mouse IgG (Jackson ImmunoResearch). ELISAs were developed with 1-Step TurboTMB-ELISA substrate (Pierce), and Absorbance at 450nm was read with a Tecan Sunrise plate reader.

DC maturation in culture

For in vitro maturation, splenocytes were cultured overnight in complete RPMI at ~8×106 cells/ml. In some experiments the 10% FBS in the RPMI was replaced with either 10% mouse serum from a non-treated mice, or 10% mouse serum from a mouse inflamed with bacterial lysate for 16 hr. For adoptive transfer experiments, spleenocytes cultured overnight to mature the DCs then intravenously injected into CD45.1 mice that we either non-treated or inflamed with bacterial lysate for 16 hr. (1.0×109 spleenocytes per mouse). After various amounts of time, 4mg GFP-OT were injected, and 30 minutes later, spleeocytes were harvested and GFP capture by the DC was analyzed by flow cytomtry as compared to a similarly treated mouse not injected with GFP. The endogenous DCs were discriminated from the transferred DCs by CD45.1 and CD45.2 staining.

Flow cytometry

Cells were pre-incubated with 10 μg/ml 2.4G2 mAb (Bio Express) for 15 min at 4°C in PBE, incubated with mAb conjugates for 30 min at 4°C and resuspended in PBE with 0.5ug/ml 7-aminoactinomycin-D (Invitrogen) 10 min before analysis. All samples were gated on live cells by scatter and 7-aminoactinomycin-D exclusion. Data was collected on a FACSCanto (BD) and analyzed with FlowJo software (TreeStar). Antibodies: PE or APC-Alexa750-CD8a (53-6.7), PE-Cy7 CD11c (N418), APCAlexa750-CD45.1(A20), Pacific Blue or APC-CD45.2 (104), APC-CD4 (GK1.5), FITC-Ly-6C (HK1.4), Alexa488, APC, or PE or APC-CD86 (GL-1) and the corresponding isotype control (Rat IgG2a), PE or APC-CD80 (16-10A1) and corresponding isotype control (Armenian Hamster IgG), PE, APC, or Pacific Blue conjugated-IA/E (M5/114.15.2) and corresponding-isotype control (Rat IgG2b), Alexa647-H2Kb (AF6-88.5) and Alexa647-isotype control (mouse IgG2a), Alexa488-CD40 (HM40-3) and Alexa488-isotype control (Armenian Hamster IgM), Alexa488 or APC-CCR7(4B12) and corresponding-isotype control (Rat IgG2a), were purchased from eBioscience or Biolegend.

Confocal Microscopy

Sorted DCs were attached to Alcian Blue 8GX (Sigma) coated cover slips by centrifugation at 100xg for 1 min and incubation at 37°C for 5 min. All subsequent steps were at room temperature. Cells were fixed in 2% PFA in PBS for 30 min, permeabilized with 0.1% Triton X-100 in PBSfor 5 min, and blocked with 5% goat serum and 10 μg/ml anti FcR-mAb (2.4G2) for 30 min. DCs were stained with anti-CD11c (N418, eBioscience) followed by Rhodamine Red-X conjugated goat anti-Armenian Hamster (Jackson Immunoresearch), Pacific Blue conjugated anti-IA/E (M5/114.15.3, Biolegend), and anti-GFP (rabbit polyclonal serum) followed by Alexa488 conjugated goat anti-rabbit (Invitrogen). Cover slips were mounted with Prolong Gold (Invitrogen) and imaged with a Zeiss Plan Apochromat 63x 1.4N.A. objective on a Zeiss LSM510 microscope.

RESULTS

DCs matured in vivo continue to capture and present soluble antigens

Systemic DC maturation was induced in mice with the TLR agonists LPS or CpG DNA. Injection of either inflammatory stimuli (LPS or CpG) resulted in systemic DC maturation evidenced by the uniform up-regulation of surface markers characteristic of maturation on spleen DCs (Figure 1A). We next compared the capacity of immature DCs (in control, non-inflamed mice) and DCs matured in vivo (in mice treated with either LPS or GpG) to internalize and present soluble antigens. Control mice or mice that had been previously inflamed (in which mature DCs had been generated and accumulated for 16 hr as described in Figure 1B) were injected intravenously with ovalbumin (OVA). The antigen (OVA) was then allowed to circulate in the mice for 30 minutes, a period much shorter than the time needed for conversion of immature into mature DCs, assuring that the phenotype of the DCs did not change during their brief exposure to the injected antigen. After this 30-minute antigen pulse, spleens were harvested and immature DCs from control mice or mature DCs from inflamed mice were isolated by cell sorting. The method used to isolate mature DCs removed residual immature or incompletely mature DCs present in the sample (Supplementary Figure 1). The isolated DCs were then assayed for their capacity to present the antigen they may have captured in vivo (Figure 1B).

Figure 1.

Figure 1

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DCs matured in vivo with LPS or CpG present subsequently encountered soluble antigen. A. Analysis by Flow cytometry of surface expression of maturation markers on spleen DC from non-treated mice (solid line), mice treated with LPS 16 hours prior (dotted line), and mice treated with CpG-B 16 hours prior (dashed line). Shaded histograms show staining obtained with isotype controls. B. Experimental Scheme for antigen presentation assays. C. Immature DCs from non-treated (NTx) mice, mature DCs from CpG inflamed mice or mature DCs from LPS inflamed mice were purified (as described in supplementary figure 1) 30 minutes after injection with 1mg OVA, and co-cultured with CFSE-labeled OT-I or OT-II T-cells over a range of T-cell/DC ratios. After 60h, antigen presentation was assessed by flow cytometric analysis of CFSE-dilution to measure T-cell proliferation. (Flow cytometry data shown is T-cell/DC ratio of 1/1) DCs purified from mice not injected with antigen were similarly analyzed. D. Quantification of T-cell proliferation as described in B induced by immature DCs from non-treated mice (Triangles), mature DCs from LPS treated mice (squares), or mature DCs from CpG treated mice (circles).

As expected, immature DCs isolated from control mice (NTx-immature) were able to stimulate OT-I CD8+ or OT-II CD4+ T-cells following injection of OVA, indicating that the DCs had captured antigen in vivo and presented the internalized antigen to T-cells (Figure 1C,D). Surprisingly, mature DCs isolated from OVA injected mice that had been previously inflamed with LPS or CpG were also able to stimulate both CD8+ OT-I and CD4+ OT-II T-cells similarly to immature DCs isolated from control mice (Figure 1C,D). These results suggested that DCs matured in vivo retained their capacity for antigen capture and its subsequent presentation to T-cells.

The maintenance of antigen capture by DCs matured in vivo is not limited to a specific TLR-ligand

To determine if our findings were specific to the individual TLR ligands utilized, we decided to utilize a whole cell lysate of E. coli, providing a natural mix of microbial products that stimulate multiple TLRs mimicking the exposure to microbes (30, 31). Injection of mice with this bacterial lysate (BL) also induced systemic maturation of both spleen DCs (Figure 2A) and lymph node DCs (Figure 2B). These changes were also accompanied by the characteristic redistribution of MHC-II to the cell surface of mature DCs (Supplementary Figure 2). Similar to our results obtained with LPS- and CpG-matured DCs, mature DCs from mice inflamed with BL continued to present OVA captured briefly after intravenous injection (Fig 2C,D and Supplementary Figure 3A). A similar result was observed after injection of a lower dose of OVA (Figure 2E and Supplementary Figure 3A). We also verified that OT-I T-cells were stimulated with similar efficacy to produce γ-IFN (Figure 2F) and develop cytotoxic effector activity (Figure 2G) by spleen DCs that were either immature or mature at the time of antigen capture.

Figure 2.

Figure 2

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DCs matured in vivo with bacterial lysate (BL) present subsequently encountered soluble antigen. A. Analysis by flow cytometry of surface expression of maturation markers on spleen DC from non-treated mice (solid line) or mice treated with BL 16 hours prior (dashed line). Shaded histograms show staining obtained with isotype controls. B Analysis as in A, but on lymph node DCs. C. Immature DCs from non-treated (NTx) mice or mature DCs from BL inflamed mice were purified (as described in supplementary figure 1) 30 minutes after injection with 2.0mg OVA, and analyzed for antigen presentation as decribed in figure 1C. D. Quantification of T-cell proliferation as described in C induced by immature DCs from non-treated mice (Triangles), or mature DCs from bacterial lysate treated mice (squares). E. Similar to experiments in B and C, except using 0.5mg OVA injections. Additional flow cytometry data is shown in Supplementary Figure 3A. B. Similar to experiments in figure 2D,E but after 60 hours the supernatants were harvested and used to measure IFN-γ released by the OT-I T-cells stimulated by immature DCs from non-treated mice (Triangles), or mature DCs from bacterial lysate treated mice (squares). C. Similar to experiments in figure 2D,E but after 60 hours, the OT-I T-cells stimulated by immature DCs from non-treated mice (Triangles), or mature DCs from bacterial lysate treated mice (squares) were assessed for cytotoxic function as described in the methods section.

Given that the mannose receptor has been reported to contribute to the internalization of OVA (32, 33), we extended our experiments by using a soluble chimeric GFP (herein referred to as GFP-OT, Supplementary Figure 4) containing MHC-I and MHC-II restricted epitopes recognized by CD8+ OT-I and CD4+ OT-II transgenic T-cells. This GFP-OT protein is devoid of carbohydrates and therefore unlikely to be internalized by lectin-like receptors. Injection of GFP-OT gave results similar to injection of OVA at two different antigen doses (Fig 3 & Supplementary Figure 3B), further supporting the retention of endocytic activity by DCs matured in vivo.

Figure 3.

Figure 3

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DCs matured in vivo with bacterial lysate (BL) present subsequently encountered GFP-OT antigen. A. Similar to experiments described in figure 2C, but using 2.0mg GFP-OT instead of 2.0mg OVA. B. Quantification of T-cell proliferation as described in A induced by immature DCs from non-treated mice (Triangles), or mature DCs from bacterial lysate treated mice (squares). C. Similar to experiments in A and B, except using 0.5mg GFP-OT injections. Complete flow cytometry data is shown in Supplementary Figure 3B.

We verified the maturation status of the DCs did not change during the isolation procedure and that the quantity and types of DCs present in the samples did not differ or reflect the contribution of monocyte-derived inflammatory DCs (Supplementary figure 5). We also wanted to verify that there was no carry-over of antigen from the DC isolation procedures or release of antigen from these purified DCs that might have been subsequently re-captured during the assay and presented to T-cells. For this, we utilized DCs purified from C3H mice, which cannot directly stimulate OT-I or OT-II T-cell proliferation due to MHC haplotype mismatch. DCs purified from C3H Mice injected with OVA did not induce T-cell proliferation, even in the presence of B6 DCs which would be able to present any released antigen (Data not shown), indicating that antigen is not released from the isolated DCs during the assay, and therefore all T-cell stimulation observed (Figures 1,2,3 and Supplementary Figure 3) was due to presentation of antigen internalized in vivo.

Presentation of antigens encountered in vivo by mature DCs is due maintenance of their capacity to internalize soluble antigen

The results of the antigen presentation experiments (Fig. 1,2,3) are compatible with different scenarios. First, these results might be due to the extracellular loading of peptides on immature and/or mature DCs, bypassing the need for internalization and processing. We ruled this out by assaying the presentation of the injected antigen by B-cells, which capture and present OVA protein very inefficiently, but can present peptides loaded extra-cellularly. B-cells isolated from mice injected intravenously with OVA-peptide were loaded it extra-cellularly and were able to induce OT-I T-cell proliferation in vitro, while B-cell from OVA injected mice did not (Supplementary Figure 6). These results indicate that in the experiments described above (Fig. 1,2,3), OVA injections did not result in detectable release of peptides that could be loaded extracellularly on MHC molecules.

Alternatively, our antigen presentation results could reflect extremely efficient processing and presentation of trace amounts of antigen that might still have been captured by most of the DCs matured in vivo following a significant but incomplete down-regulation of antigen uptake. Finally, and in contrast to prevailing views, most DCs matured in vivo could maintain the capacity to internalize antigens at a level similar to immature DCs. Because these two possibilities imply a different pattern of antigen capture by mature DCs, we evaluated directly the endocytic capacity of in vivo matured DCs by assessing their internalization in situ of intravenously injected soluble GFP-OT (Figure 4). We found that BL-mature DCs showed a similar capacity to capture intravenously administered GFP-OT antigen as immature DCs (Figure 4B), indicating that DCs matured in vivo maintain a robust capacity to internalize soluble antigens. The internalization of GFP-OT in vivo was further confirmed by microscopy. In immature DCs purified from control mice, internalized GFP-OT was present in lysosomal compartments that also showed the accumulation of MHC-II (Figure 4C) characteristic of immature DCs (18, 19). Mature DCs purified from mice treated with bacterial lysate also exhibited intracellular GFP-OT, but MHC-II accumulated at the cell surface (Figure 4C), as expected for mature DCs (34-36).

Figure 4.

Figure 4

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DCs matured in vivo internalize soluble antigens in situ A. Experimental scheme B. Control mice (NTx), or mice inflamed with bacterial lysate (BL) were intravenously injected with GFP-OT (open histogram), and 30 minutes later antigen capture by immature or mature DCs was assessed by flow cytometry as compared to DCs from a similarly pre-treated mouse not injected with antigen (filled histogram). C. Immature or mature DCs purified from mice in B were analyzed by confocal microscopy to assess antigen internalization. Scale bars: 5 μm.

Comparison of DCs matured in vivo to DCs matured in vitro

Since it is has been widely demonstrated that DCs shut down antigen capture upon maturation in vitro (2, 3, 9-19), we wanted to directly compare DCs matured in vivo to DCs matured in culture. Spleen DCs matured in vitro showed up-regulation of maturation markers was similar to that of DCs matured in vivo (Figure 5A). In the same assays where in vivo matured DCs were able to capture antigen, spleen DCs matured in vitro did not (Figure 5B). This difference was not due to factors (such as cytokines) present in the serum of inflamed mice, because the addition of mouse serum to the maturation cultures did not rescue antigen capture (Figure 5C). Furthermore, the shut down of antigen capture could not be reversed if DCs matured in culture were adoptively transferred back into a mouse and directly compared to the endogenous DCs that had been matured in vivo (Figure 5D). These results emphasize the differences in endocytic capacity between DCs matured in vivo and those matured in culture.

Figure 5.

Figure 5

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The antigen capture observed by DCs matured in vivo cannot be recapitulated with DCs matured in vitro. A. Analysis by Flow cytometry of surface expression of maturation markers on spleen DC from non-treated mice (solid line), spleen DCs form mice treated with Bacterial lysate (BL) for 16 hours (dotted line), or spleen DCs form untreated mice that were cultured for 16 hours (dashed line). Shaded histograms show staining obtained with isotype control. B. Top: Experimental scheme. Bottom: Immature DCs from non-treated (NTx), mature DCs from BL inflamed mice, or DCs matured in culture (in vitro mature) were incubated with GFP-OT for 30 minutes in vitro at 37°C (solid line), or at 0°C (shaded; to measure background) before analysis of antigen internalization by flow cytometry. C. Similar to experiments in B, except with DCs matured in vitro in media made with mouse serum from either non-treated or bacterial lysate inflamed mice, or fetal bovine serum (FBS). D. Top: Experimental scheme. Bottom: CD45.2 DCs were matured in vitro, and then adoptively transferred back into either non-treated or bacterial lysate inflamed CD45.1 mice. After a 2 hour period to allow the in vitro matured DCs to re-acclimate with the in vivo environment, GFP-OT was injected intravenously, and 30 minutes later antigen capture was assessed by the endogenous immature DCs in the non-treated mice and the endogenous mature DCs in the bacterial lysate mice, and compared to the transferred in vitro matured DCs in the same mouse.

DCs matured in vivo can capture and present antigen to T-cells in situ

We sought further direct in vivo evidence that DCs matured in vivo are able to internalize, process and present antigen to T-cells in situ. As a first test, we adoptively transferred CD8+ OT-I and CD4+ OT-II T-cells into mice, and 16 hours after the induction of DC maturation with bacterial lysate, mice were injected with soluble antigen (Figure 6A). We observed a similar extent of antigen-specific OT-I and OT-II T-cell proliferation regardless of whether most of the DCs were immature (control mice) or mature (bacterial lysate inflamed mice) at the time of antigen delivery (Figure 6A). Such antigen presentation observed when most of the DCs were either immature or mature at the time of antigen capture resulted in a similar adaptive immune response as indicated by anti-OVA IgG titers (Supplementary Figure 7).

Figure 6.

Figure 6

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DCs matured in vivo capture soluble antigen and present it to T-cells in situ. A. Top: Experimental scheme. Bottom: Proliferation of CFSE-labeled OT-I and OT-II T-cells adoptively transferred into B6 mice that were inflamed with bacterial lysate (or control treated) prior to OVA injection. 60 hours later, spleens were harvested and antigen presentation was assessed by flow cytometric analysis of CFSE-dilution to measure T-cell proliferation. B. Top: Experimental scheme. Bottom: Purified immature DCs from control mice or mature DCs from bacterial lysate inflamed mice were transferred into BM1 mice that had been previously injected with CFSE-labeled OT-I T-cells. These mice were then injected with OVA, and 60 hours later spleens were harvested and antigen presentation was assessed by flow cytometric analysis of CFSE-dilution to measure proliferation of the adoptively transferred OT-I T-cells. C. Similar to B, but mature or immature spleen DCs were transferred into Abb (MHC-II deficient) mice that had received CFSE-labeled OT-II T-cells.

To rule out the possibility that these results (Figure 6A) are exclusively due to residual immature DCs still present in the inflamed mice, we adoptively transferred OT-I T-cells into BM1 mice, which due to point mutations in H2-Kb are unable to present OVA-derived peptides to OT-I T-cells (25, 26). As expected, when these mice were injected with OVA, no proliferation of the transferred OT-I T-cell was detected (Figure 6B). However, when purified immature DCs from a control (non-treated) wild-type B6 mouse were adoptively transferred into BM1 mice prior to the injection of OVA, proliferation of the transferred OT-I T-cells was observed (Figure 6B). The same result was obtained when we transferred mature DCs isolated from inflamed wild-type B6 mice into BM1 recipients (Figure 6B). Because the endogenous DCs in the BM1 mouse cannot present antigen to OT-I T-cells, these results confirm that the transferred BL-mature DCs were able to capture, process, and present soluble antigen in situ. We conducted a similar experiment to assess presentation on MHC-II. We found that to OT-II T-cells that had been adoptively transferred into Abb (MHC-II KO) mice responded similarly when mature or immature DCs were adoptively transferred (Figure 6C) confirming that spleen DCs matured in vivo can capture and present antigen on both MHC-I and MHC-II.

DISCUSSION

Our findings that mature DCs can capture and present antigens to CD8+ and CD4+ T-cells provide interesting new perspectives on antigen sampling during inflammation. Our results indicate that antigen capture is sustained by mature DCs. Such capacity for antigen uptake by mature DCs may be important during infections, when the continuous capture and presentation of antigens by all the available DCs (regardless of their maturation status) that have access to infected tissues and/or to microbial pathogens might offer a greater opportunity to contribute to the stimulation of adaptive immunity. Although our studies relied on a experimentally synchronized populations of mature DCs from mouse spleen, large numbers of mature DCs have been described during viral infections, bacterial infections (41, 42) and autoimmune disorders (43, 44). Our results suggest that populations of mature DCs present under various inflammatory conditions may play a role in antigen capture and presentation without necessarily relying on the generation of additional immature DC precursors.

Previous studies have described impaired (9) or enhanced (45) internalization and presentation of cell-associated antigens by DCs in mice exposed to TLR ligands. It will be important to elucidate the effects of natural infections or inflammatory process on the uptake of soluble and cell-associated antigens by DCs. It will also be crucial to discern the type(s) of T-cell priming and immunomodulatory response(s) to antigens captured by mature DC, given the increasingly appreciated capacity to mature DCs to stimulate T-regs. Also, mature DCs may be abundant in an environment rich infected apoptotic cells and their capacity to internalize and present antigens can contribute to induction of Th17 T-cell development (47).

Our findings may also relate to the mechanism of action of adjuvants that induce DC maturation. Mature DCs may be present at sites of vaccination, in an environment where inflammation and TLR-ligand stimulation may be prevalent. Thereofore, antigen capture by mature DCs at (or near) vaccination sites may contribute to T-cell stimulation without relying on a constant supply of immature DCs that need to be subsequently activated by the vaccine formulations. The continued antigen capture by DCs seems to be in line with the behavior of macrophages, which also maintain or enhance antigen capture upon activation by inflammatory stimuli (48).

Additional questions remain regarding the potentially different pathways of processing and presentation of antigens captured by mature or immature DCs. While immature DCs load antigen onto MHC-II in specialized intracellular compartments, the redistribution of MHC-II to the cell surface upon maturation (Figure 4C) (34-36, 49, 50) suggests that antigens internalized by mature DCs are likely to be loaded onto MHC-II that is recycled from the plasma membrane, as recently proposed for antigens internalized by recptor-mediated uptake by bone marrow-derived DCs (28). This process may benefit from the enhanced antigen processing observed upon TLR engagement and DC maturation (51, 52). It will also be interesting to evaluate the role of different routes of cross-presentation (7, 53-55) and their modulation by signals that also induce DC maturation (56, 57). A more detailed understanding of the contribution of small GTPases (11, 12, 58), cytoskeletal rearrangements (10), and the expression of different endocytic receptors in the capture, processing and presentation of antigen by DCs at different stages of maturation will provide a better understanding of the role of DCs in regulating T-cell responses under resting and inflammatory settings.

Supplementary Material

1

ACKNOWLEDGEMENTS

We would like to thank Peter Lopez and Gelo de la Cruz for assistance with cell sorting.

This work was supported by the NIH-NIA F30AG032190 (S.B.D.), the American Heart Association 0435251N (E.S.T), the American Cancer Society RSG-07-01-LIB and the Cancer Research Institute 63-1-7125 (E.S.T).

Footnotes

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