| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
* Department of Internal Medicine, Division of Clinical Pharmacology, Ludwig-Maximilians-University, Munich, Germany;
Institute of Immunology, University of Heidelberg, Heidelberg, Germany; and
Division of Clinical Onco-immunology, Ludwig Institute for Cancer Research, Center Hospitalier Universitaire Vaudois (CHUV), Lausanne, Switzerland
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and IFN-
) in plasmacytoid dendritic cells. In the present study, we examined the effects of CpG-A on human primary monocytes. In PBMC stimulated with CpG-A and GM-CSF, monocytes showed excellent survival, increased in size and granularity, and within 3 days developed a dendritic cell-like phenotype that was characterized by down-regulation of CD14, partial up-regulation of CCR7, and an increased surface expression of costimulatory and Ag-presenting molecules. This effect could be inhibited by a combination of blocking Abs to type I IFN, and no such CpG-A-induced changes were observed in purified monocytes. Although IL-12 production by this dendritic cell-like phenotype required additional stimulation with CD40 ligand, this cell type spontaneously up-regulated IL-15 expression. Consistent with the known effect of IL-15 on effector and memory CD8 T cells, the frequency of CCR7-/CD45RA- CD8 T cells was selectively increased in allogeneic T cell assays. Furthermore, this dendritic cell type was more potent to support both the generation and the IFN-
production of autologous influenza matrix peptide-specific memory CD8 T cells as compared with dendritic cells generated in the presence of GM-CSF and IL-4. In conclusion, monocytes exposed to the cytokine milieu provided by CpG-A rapidly develop a dendritic cell-like phenotype that is well equipped to support CD8 T cell responses. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The CpG motif recognized by the murine and human immune system differs (4, 5). Based on the activation of primary B cells, a CpG motif was identified with potent activity in the human immune system (4). Nuclease-stable phosphorothioate ODN4 were developed that proved to be excellent vaccine adjuvants in primates (CpG-B; prototype ODN 2006 (6, 7, 8)). Besides B cells, the plasmacytoid dendritic cell (PDC) represents a primary target cell of CpG ODN in humans (9, 10, 11, 12, 13, 14). The PDC is characterized by the production of extremely large amounts of type I IFN upon viral infection (15, 16). It has been reported that CpG-B ODN promote survival, activation, and maturation of PDC, but that the induction of type I IFN production was weak as compared with viruses (10, 11, 12, 13). Based on the ability to stimulate type I IFN in PDC, a novel type of CpG ODN was identified (CpG-A; prototype ODN 2216) that induces maximal amounts of IFN- and IFN- in PDC (400 ng/ml IFN- in the supernatant; 5 pg/single PDC (13)) and in this regard seems to mimick viral infection.
Toll-like receptor 9 (TLR9) is critically involved in the recognition of CpG motifs (5, 17, 18). Although considerable levels of TLR9 were found in B cells and PDC, TLR9 is weak or absent in monocytes and peripheral blood myeloid dendritic cells (DC) (12, 14). Consistent with TLR9 expression, monocyte-derived DC and myeloid DC are not sensitive to CpG ODN (5, 10, 12). Although TLR9 expression is weak in monocytes (14), monocytes have been described to respond to bacterial DNA and CpG ODN (11, 19, 20). Sensitivity of monocytes toward CpG motifs may change while monocytes enter distinct differentiation pathways depending on growth factors and cytokines to which they are exposed. There are several examples of different differentiation pathways that monocytes are capable to enter. In the presence of receptor activator of NF-
B ligand and M-CSF, multinucleated osteoclasts develop (21). Although GM-CSF alone leads to macrophage development, GM-CSF together with IFN- drives the formation of multinucleated Langhans’ giant cells (22). DC develop in the presence of GM-CSF and IL-4 (23) or during transendothelial migration (24).
Type I IFNs are an important link between innate and acquired immunity, and mediate a variety of immunoregulatory effects (25). Type I IFN have been demonstrated to modulate the development and the phenotype of monocyte-derived DC (26, 27, 28, 29, 30). Furthermore, type I IFN strongly activate NK cells, NKT cells (31), and 
T cells (32), and preferentially promote the expansion of memory CD8 T cells in vivo (33), an effect that is mimicked by IL-15 in vivo and in vitro (34). In addition, type I IFN directly prevent apoptosis of Ag-activated T cells in vitro (35). Direct type I IFN-mediated inhibition of Ag receptor-mediated apoptosis also occurs in B cells. In vivo, type I IFN lead to enhanced humoral immunity and to isotype switching of B cells (36).
Teleologically, it would make sense that the set of immune responses induced by a certain pathogen includes the generation of a DC phenotype that specifically promotes the type of T cell function appropriate to overcome the corresponding type of infection. In the present study, we examined the impact of a IFN--inducing CpG ODN (CpG-A, ODN 2216) on monocyte differentiation and DC development. We found that upon stimulation with this type of CpG ODN in the context of PBMC, monocytes acquired a DC phenotype that was characterized by spontaneous expression of IL-15, but not IL-12, and that preferentially supported CD8 T cells.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
RPMI 1640 culture medium (Biochrom, Berlin, Germany) supplemented with 5% (v/v) pooled human AB serum (BioWhittaker, Walkersville, MD), 1.5 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (Sigma-Aldrich, Munich, Germany) was used throughout (culture medium). All compounds were purchased endotoxin tested. Completely and partially phosphorothioate-modified ODN were provided by Coley Pharmaceutical Group (Wellesley, MA) (small letters, phosphorothioate linkage; capital letters, phosphodiester linkage 3' of the base; bold, CpG dinucleotides): ODN 2006, 5'-tcgtcgttttgtcgttttgtcgtt-3' (7); ODN 2216, 5'-ggGGGACGATCGTCgggggG-3'; ODN 2243, 5'-ggGGGAGCATGCTCgggggG-3' (GC control to ODN 2216) (13). ODN were used at a concentration of 3 µg/ml. No endotoxin could be detected in ODN preparations (<0.03 EU/ml; Limulus amebocyte lysate assay; BioWhittaker). GM-CSF (Novartis, Basel, Switzerland) was used at 1000 U/ml. IL-4 (PeproTech, Rocky Hill, NJ) and IFN-2a (PBL Biomedical Laboratories, New Brunswick, NJ) were both used at 500 U/ml. TNF- (R&D Systems, Wiesbaden, Germany; 1000 U/ml), PGE2 (Sigma-Aldrich; 1000 U/ml), IL-1 (Strathmann Biotech; 10 ng/ml), IL-6 (R&D Systems, Hamburg, Germany; 10 ng/ml), IFN- (Boehringer Mannheim, Indianapolis, IN; 1000 U/ml), poly(I:C) (Sigma-Aldrich; 12.5 µg/ml), and CD40 ligand (CD40L) (irradiated human CD40L transgenic BHK cells used in a ratio of 1:10; kindly provided by H. Engelmann, Munich, Germany) were used as maturation stimuli, as indicated. For blocking the effect of type I IFN, a mixture of 5000 neutralizing U/ml rabbit anti-human IFN- Ab, 2000 NU/ml rabbit anti-human IFN- Ab, and 20 µg/ml mouse anti-human IFN-/ receptor mAb IFN-R1 was used (PBL Biomedical Laboratories). TNF was neutralized by anti-TNF mAb (Endogen, Woburn, MA). CpG-conditioned medium (CpG-CM) was prepared by collecting the cell-free supernatant of PBMC (3 x 106/ml) after stimulation with CpG ODN (3 µg/ml) for 24 h.
Preparation and culture of cells
Human PBMC were isolated from blood or buffy coats of healthy volunteers, as described. For preparation of highly purified untouched monocytes free of PDC, PBMC were first depleted of PDC by direct magnetic labeling with anti-blood DC Ag (BDCA)-4-coupled magnetic beads (Milteny Biotec, Bergisch Gladbach, Germany). The remaining cells were then depleted of T cells, NK cells, B cells, and basophils using the monocyte isolation kit from Miltenyi Biotec, as described by the manufacturer. Untouched T cells (CD4+ and CD8+) were isolated from PBMC by depleting other cell types (pan T cell isolation kit; Miltenyi Biotec). CD8+ T cells were isolated using a positive selection with anti-CD8 microbeads (Miltenyi Biotec). After preparation of the different cell populations, viability was determined by trypan blue exclusion (96–99% viable).
PBMC were suspended in supplemented culture medium at 2 x 106 cells/ml and incubated with GM-CSF alone or together with CpG ODN, IFN-2a, or IL-4 for 3–6 days, as indicated. For blocking experiments, neutralizing Abs were added at the beginning of the culture. Purified monocytes were cultured at 1 x 106/ml and incubated with GM-CSF, GM-CSF/IFN-, or GM-CSF/IL-4. For some experiments, monocytes were resuspended in CpG-CM at 1 x 106 cells/ml and cultured with GM-CSF. Immature DC were generated, as described, by culturing isolated monocytes (1 x 106 cells/ml) with GM-CSF and IL-4 for 3–5 days; for maturation, maturation stimuli together with fresh medium containing GM-CSF and IL-4 were added for 48 h. Alternatively, two-thirds of the medium were replaced by CpG-CM, and cells were cultured with fresh GM-CSF and IL-4 for 48 h. At the indicated time points, supernatants were collected for detection of cytokines, and cells were harvested by vigorous pipetting with ice-cold PBS (no remaining cells on the cell culture plate) for flow cytometric analysis.
Flow cytometry
Surface Ag staining was performed, as previously described (37). Fluorescence-labeled mAbs against CD80, CD86, CD40, CD54, HLA-DR, MHC I, CD83, CD11c, CD4, CD8, CD45RA, and appropriate isotype control Abs were purchased from BD PharMingen (San Diego, CA). Anti-CD14 mAb and the matching isotype control mAb were purchased from Immunotech (Marseille, France). CCR7 expression was detected by incubation with anti-CCR7 mAb (rat IgG2a, clone 3D12; kindly provided by R. Förster), followed by biotinylated anti-rat IgG2a mAb (clone RG7/1.30) and streptavidin-APC (both from BD PharMingen, Heidelberg, Germany). PE-coupled HLA-A2/Flu matrix58–66 tetramers (kindly provided by P. Guillaume, Ludwig Institute for Cancer Research, Lausanne branch, Epalinges, Switzerland) were synthesized, as previously described (38). For detection of intracellular cytokines, T cells were first labeled with FITC-labeled anti-CD8 and PerCP-labeled anti-CD3 mAb, then fixed, permeabilized (Fix and Perm Kit; Caltag, Burlingame, CA), and stained with PE-labeled anti-IFN- or isotype control Abs (BD PharMingen). Flow cytometric data were acquired on a BD Biosciences (Heidelberg, Germany) FACSCalibur equipped with two lasers (excitation at 488- and 635-nm wavelength). Analysis was performed on viable cells within a morphologic gate (forward light scatter (FSC), side light scatter (SSC), >97% of viable cells, as confirmed by propidium iodide staining). For analysis of monocyte-derived cells within PBMC, a gate was set on large granular cells (high FSC and SSC) that expressed the myeloid marker CD11c. For the measurement of cell yield and survival of monocyte-derived cells within PBMC, 100 µl of the resuspended PBMC was harvested and stained with FITC-labeled anti-CD11c mAb and TO-PRO-3 iodide (Molecular Probes, Eugene, OR) for dead cell exclusion. After staining, cells were resuspended in 200 µl, and the absolute number of live CD11c+ cells per 100 µl aspirated volume was determined by flow cytometry and used to calculate the concentration of cells in the PBMC suspension. Data were analyzed using CellQuest (BD Biosciences) or FlowJo software (version 2.5.1; Tree Star, Stanford, CA). Gates were set according to isotype control samples.
Detection of cytokines
For the detection of IL-12 and IL-10 in the cell-free supernatant, the human IL-12 (p40/p70) ELISA, range 62.5–2000 pg/ml (Bender Med Systems, Vienna, Austria), and the human IL-10 OptEIA, range 7.8–500 pg/ml (BD Biosciences), were used. For detection of IFN-, human IFN- multispecies ELISA, range 100-5000 pg/ml (PBL Biomedical Laboratories), was used.
Real time RT-PCR
The protocol used for real time RT PCR was described in detail earlier (14). Briefly, cells were lysed, and RNA was extracted using the total RNA isolation kit (High Pure; RAS, Mannheim, Germany). An aliquot of 8.2 µl RNA was reverse transcribed using avian myeloblastosis virus-reverse transcriptase and oligo(dT) as primer (First Strand cDNA Synthesis Kit; Roche, Mannheim, Germany). The obtained cDNA was diluted 1/25 with water, and 10 µl was used for amplification. An IL-15-specific primer set optimized for the LightCycler (RAS) was developed by Search-LC GmbH (Heidelberg, Germany) and purchased from Search-LC. The PCR was performed with the LightCycler FastStart DNA Sybr GreenI kit (RAS), according to the protocol provided in the parameter-specific kits. The copy number was calculated from a standard curve, obtained by plotting known input concentrations of four different plasmids at log dilutions to the PCR cycle number (CP) at which the detected fluorescence intensity reaches a fixed value. Using over 300 data points, the actual copy number per microliter of cDNA was calculated as follows: X = e(-0.6553*CP + 20.62). The data of two independent analyses for each sample and parameter were averaged. The copy number was normalized by the housekeeping gene cyclophilin B, and is presented as number of transcripts per 103 copies of cyclophilin B.
T cell assays
To examine the effect of different monocyte-derived cell types on CD4 and CD8 T cell subsets, purified monocytes were incubated with GM-CSF alone or in combination with CpG-CM, IFN-2a, or IL-4. After 3 days, cells were harvested, washed, counted, and used as stimulator cells in an allogeneic MLR. Pan T cells from a different donor were isolated. For measurement of T cell proliferation, T cells were stained with CFSE (Molecular Probes), as described (39). Pan T cells were coincubated with 1 x 104 monocyte-derived cells/well in 96-well round-bottom plates. After 6 days of coculture, the percentage of proliferating T cells is determined by a decrease in CFSE staining on CD4 and CD8 T cells, respectively. Expression of CD45RA and CCR7 was analyzed on CD8 and CD4 T cells. For the expansion of influenza-specific T cells, monocytes of HLA-A2-positive donors were incubated for 3 days with GM-CSF and IL-4 or GM-CSF and ODN 2216-conditioned medium. After 3 days, cells were harvested and washed, and 1 x 105 monocyte-derived cells were pulsed with 0.5 µM of the immunodominant HLA-A2-restricted peptide of the influenza matrix protein (GILGFVFTL referred to as Flu matrix58–66) and used to stimulate 1 x 106 autologous CD8 T cells. After 10 days, the expansion of Ag-specific CD8+ T cells was analyzed by flow cytometry using PE-coupled HLA-A2/Flu matrix58–66 tetramers. For the assessment of Ag-specific IFN- production, cells were harvested after 10 days, washed once, and restimulated in 96-well round-bottom culture plates in 200 µl medium with 10 µM of their cognate peptide or the HIV-pol476–484 peptide as a control. After 2-h incubation at 37°C, 1 µg/ml brefeldin A (Sigma-Aldrich) was added. After an additional 4-h incubation at 37°C, cells were harvested, and intracellular cytokine staining was performed.
Statistical analysis
Data are expressed as means ± SEM, unless otherwise indicated. Statistical significance of differences was determined by the paired two-tailed Student t test (p < 0.05). Statistical analyses were performed using Stat-View 4.51 software (Abacus Concepts, Calabasas, CA).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Our previous studies indicated that within PBMC, human monocytes are activated by bacterial DNA or CpG ODN to up-regulate ICAM-1 (CD54) and to produce inflammatory cytokines (40). We hypothesized that different types of CpG ODN may differ in their ability to impact on monocyte activation and differentiation. We screened a series of CpG ODN sequences for their activity to stimulate the expression of costimulatory molecules on monocytes. Among the CpG ODN tested, the recently described CpG-A sequence ODN 2216 was most active to up-regulate CD80 expression on monocytes within PBMC (data not shown). ODN 2216 not only activated monocytes, but also partially down-regulated CD14 expression, suggesting that monocytes might enter a differentiation pathway toward DC development (not in figure). Down-regulation of CD14 by ODN 2216 was more pronounced in the presence of GM-CSF. To evaluate the potency of ODN 2216 to promote DC development under conditions comparable to other DC protocols from the literature, PBMC were incubated with GM-CSF alone or in combination with ODN 2216. Other conditions to generate monocyte-derived cells were GM-CSF together with rIL-4 (standard protocol for the generation of monocyte-derived DC) or GM-CSF with IFN-2a. After 3 days of culture, monocyte-derived cells were identified by FSC/SSC characteristics as large granular cells (>95% of these cells were CD11c+), and their phenotype was analyzed by flow cytometry. As shown in Fig. 1A, ODN 2216 (CpG-A) strongly induced the expression of CD80, CD40, and CD54 in monocytes as compared with GM-CSF alone (mean fluorescence intensity (MFI) values ± SEM for CD80, 79 ± 18 vs 32 ± 12; for CD40, 133 ± 30 vs 58 ± 15; for CD54, 491 ± 98 vs 249 ± 50; n = 6, p < 0.05). Up-regulation of CD86 and HLA-DR by CpG ODN was less pronounced (CD86, 101 ± 22 vs 74 ± 12; HLA-DR, 131 ± 29 vs 89 ± 23; n = 6). Activation of monocytes in the presence of GM-CSF and IFN- was similar as compared with stimulation with GM-CSF and ODN 2216. In contrast, monocytes cultured with GM-CSF and IL-4 exclusively up-regulated CD86 (148 ± 32 vs 74 ± 12; n = 6, p < 0.03). Consequently, the expression ratio of CD80 to CD86 on monocytes was higher in PBMC cultured with GM-CSF/CpG-A or GM-CSF/IFN- than with GM-CSF/IL-4. The DC maturation marker CD83 was not significantly up-regulated on monocytes with any of the stimuli used. The majority of the increase of HLA-DR, CD80, and CD86 by CpG-A was found within the first 3 days of culture (Fig. 1B). CD14 was markedly down-regulated in all conditions by day 3. Most monocytes completely lost CD14 expression after culture of PBMC with GM-CSF and IL-4, whereas monocytes cultured with GM-CSF, GM-CSF/CpG-A, or GM-CSF/IFN- still expressed low levels of CD14 (Fig. 1B, upper left panel).
|
, and for ODN 2216 alone. In contrast, the addition of IL-4 to GM-CSF did not enhance survival of monocyte-derived cell as compared with GM-CSF alone. The non-CG control ODN 2243 (CGs switched to GCs) was inactive. Furthermore, the CpG-B ODN 2006 did not significantly increase survival of monocyte-derived cells.
|
To generate pure monocyte-derived cell populations, monocytes were isolated from PBMC by depleting other cell types, including plasmacytoid DC (>97% monocytes; <0.03% PDC). Monocytes purified by this method were not sensitive to stimulation with CpG ODN 2216 (no induction of CD80, CD86, HLA-DR, CD40, CD54), indicating that there is no direct effect of CpG ODN on monocytes, which is consistent with the lack of TLR9 expression in monocytes (14) (Fig. 3A, hatched bars). When PDC depletion was omitted from the purification protocol (
1% remaining PDC in monocyte preparation), partial activation of monocytes by CpG ODN alone was observed, indicating an indirect effect mediated by PDC-derived cytokines (not shown). When purified monocytes (no contaminating PDC) were incubated in the presence of supernatant derived from PBMC stimulated with ODN 2216 (CpG-CM) with or without GM-CSF, monocytes demonstrated marked up-regulation of CD80, CD86, HLA-DR, CD40, and CD54 (Fig. 3A, filled bars; Fig. 3B, left panel) similar to monocyte-derived cells generated with GM-CSF and ODN 2216 within PBMC (compare Fig. 1A). This effect was CpG specific because supernatants of PBMC stimulated with the non-CG control ODN 2243 (CGs switched to GCs) in combination with GM-CSF were negative (Fig. 3B). Furthermore, supernatants derived from PBMC stimulated with CpG-B ODN 2006 and GM-CSF showed no such effect. In addition to costimulatory molecules and MHC II, MHC I (HLA-A/B/C) also was up-regulated by supernatants generated in the presence of ODN 2216 (CpG-A), but not CpG-B ODN 2006 nor non-CpG-containing ODN (Fig. 3B, right panel).
|
, IFN-, and IFN-/ receptor to GM-CSF/CpG-A-stimulated PBMC abrogated CpG-A-induced expression of CD80 on monocytes (Fig. 4). Neutralization of IFN- alone also markedly reduced the CpG-A-mediated activation of monocytes, whereas anti-TNF- Ab showed no significant changes (Fig. 4).
|
, but not in monocytes incubated in the presence of GM-CSF and IL-4 or with GM-CSF alone (Fig. 5).
|
In general, full maturation of monocyte-derived DC requires additional stimulation. We tested different maturation stimuli that are known to induce full maturation of monocyte-derived DC generated in the presence of GM-CSF and IL-4 (41). Monocytes were incubated with GM-CSF in combination with IL-4 or ODN 2216-conditioned medium. After 4 days, CD40L; poly(I:C); the combination of CD40L, poly(I:C), and IFN-; or a cytokine cocktail consisting of IL-1, TNF-, IL-6, and PGE2 was added as maturation stimuli. After another 2 days, the expression of surface markers and the production of IL-12 and IL-10 were examined.
In monocyte-derived cells generated in the presence of GM-CSF and ODN 2216-conditioned medium (GM-CSF/CpG-A-CM cells; Fig. 6, upper panel), elevated levels of CD80 without stimulus (medium) were further up-regulated by all maturation stimuli used. In monocyte-derived DC generated in the presence of GM-CSF and IL-4 (GM-CSF/IL-4 cells; Fig. 6, lower panel), baseline expression of CD80 was low, and the combination of CD40/IFN-/poly(I:C) or the cytokine cocktail TNF/IL-1/IL-6/PGE2 was required to achieve similarly high levels of CD80 expression as seen in GM-CSF/CpG-A-CM cells. Consistent with the literature, GM-CSF/IL-4 cells strongly up-regulated CD83 and CCR7 expression in response to maturation stimuli, while the slightly elevated baseline level of CD83 in GM-CSF/CpG-CM cells showed no or only minor further up-regulation upon stimulation. CCR7 expression in GM-CSF/CpG-A-CM cells was only increased in the presence of CD40L (CD40L alone or CD40L/IFN-/poly(I:C)). Upon stimulation with CD40L (CD40L alone or CD40L/IFN-/poly(I:C)), GM-CSF/CpG-A-CM cells produced high levels of IL-12 (>20 ng/ml with CD40L/IFN-/poly(I:C)) and considerable amounts of IL-10. IL-12 production was even higher in GM-CSF/IL-4 cells (>100 ng/ml IL-12 with CD40L/IFN-/poly(I:C)). Production of IFN- could not be detected in both cell types.
|
It has been reported that immature human DC generated from purified monocytes in the presence of GM-CSF and IL-4 are not directly activated by CpG ODN (5, 10). We examined whether these DC are sensitive to indirect activation by CpG ODN. Immature DC were generated from isolated monocytes by using a standard protocol (GM-CSF and IL-4). After 5 days, CpG-A-CM was added, and after another 2 days, surface markers were studied by flow cytometry. As shown in Fig. 7A (hatched bars), in the presence of CpG-A-CM the expression of CD80, CD86, and HLA-DR was increased (mean MFI value ± SEM of CD80, 50 ± 8 vs 24 ± 3; CD86, 103 ± 19 vs 49 ± 16; HLA-DR, 77 ± 13 vs 54 ± 22; n = 3). The level of activation was lower as compared with a standard cytokine cocktail (filled bars). Furthermore, CD83 was strongly up-regulated by the standard cytokine cocktail (filled bar, MFI 73 ± 30), but was not increased by CpG-CM as compared with control (hatched bars, MFI 23 ± 9 vs 24 ± 7; n = 3). Although CpG-CM stimulated some IL-12 production of GM-CSF/IL-4-generated DC (Fig. 7B, hatched bar), IL-12 production was weak as compared with stimulation by the cytokine cocktail (Fig. 7B, closed bar).
|
We compared the T cell stimulatory capacity of the different monocyte-derived cell types. Isolated monocytes were incubated with GM-CSF in combination with CpG-A-CM, IFN-, or IL-4 for 3 days. Monocyte-derived cell types were coincubated with CFSE-labeled CD3 T cells from a different donor. After 5 days, proliferation was determined by the intensity of CFSE staining (low CFSE staining indicates proliferation) on CD4 and CD8 T cells. As expected, proliferation of both CD4 and CD8 T cells was lowest in the presence of monocyte-derived cells generated in the presence of GM-CSF alone (Fig. 8A, left panel). Monocyte-derived cells generated in the presence of GM-CSF and IL-4 induced proliferation of CD4 T cells and a smaller number of CD8 T cells (Fig. 8, second panel from left). Monocyte-derived cells generated in the presence of GM-CSF and CpG ODN 2216 stimulated the proliferation of both CD4 and CD8 T cells (Fig. 8, third from left).
|
(Fig. 8C; mean percentage ± SEM, 45 ± 2% and 35 ± 6%, n = 4), but this difference did not reach statistical significance. Consistent with the increase in CD45RA-/CCR7- CD8 T cells, the percentage of CD45RA+/CCR7+ CD8 T cells (naive T cells) decreased (Fig. 8C). Unlike for CD8 T cells, no consistent changes of the CD45RA and CCR7 phenotype of CD4 T cells were observed (Fig. 8C).
Monocyte-derived cells generated in the presence of GM-CSF and CpG-A ODN support the expansion and IFN- production of influenza-specific memory CD8 T cells
Because GM-CSF/CpG-A-CM cells increased the frequency of CD45RA-/CCR7- CD8 T cells (so-called effector memory T cells) in an allogeneic system, we studied the ability of this cell type to support an autologous peptide-specific CD8 T cell memory response. An HLA-A2-restricted peptide from the influenza matrix protein was used as a model Ag to study a recall response in adults that have experienced influenza infections during their lifetime. Monocytes of HLA-A2-positive donors were incubated with GM-CSF and IL-4 (GM-CSF/IL-4 cells) or GM-CSF and ODN 2216-conditioned medium (GM-CSF/CpG-A-CM cells). After 3 days, monocyte-derived cells were pulsed with the HLA-A2-restricted peptide Flu matrix58–66 and used to stimulate autologous CD8 T cells. Tetramer staining revealed a frequency of 0.05–0.2% of influenza matrix peptide-specific CD8 T cells before stimulation. After 10 days of stimulation with peptide-pulsed GM-CSF/CpG-A-CM cells, a 40- to 150-fold increased frequency of influenza matrix peptide-specific CD8 T cells (7.8% of all CD8 T cells) was found (Fig. 9A). These T cells were functionally active, as revealed by intracellular IFN- staining upon restimulation with influenza matrix peptide (Fig. 9B). The background of IFN--producing cells measured by restimulation with a HLA-A2-restricted control peptide derived from HIV-pol was below 0.3% IFN-+ cells for all conditions (data not shown). GM-CSF/CpG-A-CM cells were more active than GM-CSF/IL-4 cells to support the generation of peptide-specific CD8 T cells (Fig. 9B; p < 0.05).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, may mimick a molecular pattern used by the immune system to recognize the presence of virus, and thus may lead to an immune response that is appropriate to limit viral infection.
In the present study, we focused on the effect of CpG-A on monocytes. We found that CpG-A (ODN 2216), but not CpG-B (ODN 2006), has a strong impact on monocyte differentiation and function. In the presence of GM-CSF and CpG-A, monocytes within PBMC decreased CD14 expression and rapidly developed a DC-like phenotype (CpG-A DC). The development of CpG-A DC within PBMC depended on CpG-A-induced IFN-. Purified monocytes were not sensitive to the CpG-A, but the development of CpG-A DC could be restored by the addition of PBMC-derived CpG-A-CM. CpG-A DC expressed high levels of CD40, CD54, CD80, MHC I, and MHC II without an additional stimulus. CpG-A DC rapidly up-regulated high levels of IL-15 mRNA that were maintained at lower levels throughout differentiation. Further maturation and IL-12 production of CpG-A DC required additional CD40L-mediated stimulation. Even without such further stimulation, CpG-A DC supported the proliferation of allogeneic CD8 T cells, selectively increasing the frequency of CCR7-/CD45RA- CD8 T cells over other CD8 T cell subpopulations. In the autologous setting, CpG-A DC strongly increased the expansion of influenza matrix peptide-specific memory CD8 T cells.
Comparing CpG-A DC with DC generated in the presence of GM-CSF and IL-4 (GM-CSF/IL-4 DC), an interesting feature of CpG-A DC is the spontaneous expression of high levels of costimulatory molecules and of IL-15 mRNA. This explains the high activity of CpG-A DC to promote a peptide-specific memory CD8 T cell response in the absence of a further stimulus. To achieve similar levels of the costimulatory molecule CD80, GM-CSF/IL-4 DC require an additional maturation stimulus. In the presence of such a stimulus, GM-CSF/IL-4 DC expressed higher levels of CD83, CCR7, and IL-12 than CpG-A DC. Therefore, it seems unlikely that CpG-A DC can be used instead of the gold-standard mature GM-CSF/IL-4 DC for ex vivo vaccination protocols. However, while GM-CSF/IL-4 DC require ex vivo protocols for their clinical use, CpG-A (also termed D type CpG ODN (20)) can be administered directly in vivo (42). Therefore, our study contributes to the understanding of the monocyte-differentiation pathway elicited by CpG-A rather than providing a new ex vivo DC generation protocol.
Our results on activation and differentiation of monocytes toward a DC-like phenotype by CpG-A are in agreement with recent results from the literature (20, 43, 44). Our study extends previous data by adding evidence that DC generated in the presence of this type of CpG ODN display a distinct activity regarding T cell differentiation. CpG-A DC were more potent than GM-CSF/IL-4 DC to expand peptide-specific memory T cells. Evidence for the functional activity of expanding CD8 T cells is provided by the capacity of these expanding CD8 T cells to produce IFN- upon restimulation with the corresponding peptide Ag. Elevated levels of IL-15 mRNA in CpG-A DC, as demonstrated in our study, may contribute to the activity of CpG-A DC to support memory CD8 T cells. IL-15 is known to bind to the -chain of the IL-2R, which is preferentially expressed on memory CD8 T cells (34).
Based on the expression of CCR7, which is responsible for homing in secondary lymphoid organs, and of CD45RA, T cells can be divided in CD45RA+ naive T cells, as well as in two subsets of preactivated T cells (central memory T cells, CD45RA-/CCR7+; effector memory T cells, CD45RA-/CCR7-) (45). An intriguing feature of CpG-A DC (in contrast to GM-CSF/IL-4 DC) was its ability to increase the frequency of the CCR7-/CD45RA- subset of CD8 T cells, while no such selective increase was found for CD4 T cells. It has been proposed that CCR7-/CD45RA- effector memory T cells enter peripheral tissues and secrete inflammatory cytokines or exert effector function (45). In earlier studies, it has been shown that CpG-A (ODN 2216) potently stimulates the lytic activity and IFN- production of NK cells and T cells within PBMC (32, 46). Together with the expansion of CD8 memory T cells, as shown in the present study, this type of CpG ODN seems to initiate a set of early immune responses useful to limit viral replication.
The induction of type I IFN plays an essential role in CpG ODN-mediated activation of NK cells and T cells (32), as well as in the development of CpG-A DC, as demonstrated in the present study. It is known that type I IFN are responsible for the impaired IL-12-mediated immunity during viral infections (47). Type I IFN reduce the capacity of DC to produce IL-12 in response to microbial or CD40L-mediated stimulation (27, 48). As a consequence, DC are less active to promote naive CD4 T cell proliferation and to induce IFN--producing Th1 cells (48, 49). The potential of these DC to stimulate CD8 memory T cells was not examined in these two studies.
In another study, it has been demonstrated that monocytes incubated in the presence of GM-CSF and type I IFN develop a DC-like phenotype (28). Similar to our results, in that study DC showed only partial up-regulation of CD83 expression and an advanced stage of differentiation. IL-12 and IL-15 production in DC and IFN- production in DC-stimulated T cells were not examined. Another group reported the development of a monocyte-derived DC phenotype in the presence of GM-CSF and type I IFN (30). In this study, the authors observed an increased IFN- production in the supernatant of DC-stimulated PBMC without identifying the cell population responsible for this IFN- production. Consistent with our results, in their study, DC generated in the presence of GM-CSF and type I IFN lacked spontaneous IL-12 expression, but produced high amounts of IL-15 in the absence of a maturation stimulus. In another study, it has been reported that IL-15 is also expressed in mature IL-12-competent DC (50). Regarding the function of IL-15, it has been shown that IL-15 stimulates memory-phenotype CD8 T cells, but not CD4 T cells in vitro and in vivo (34). Furthermore, IL-15 production by DC correlated with expansion of Ag-specific preactivated CD8 T cells (51). Together, these studies and our own results are in agreement with the concept that type I IFN promote the development of a monocyte-derived DC-like phenotype that exhibits impaired IL-12 function, but via expression of IL-15, selectively supports CD8 memory T cells.
Differences between these studies regarding the DC phenotype, such as the level of CD83 expression, IL-12 expression, and final maturation, may be due to the source and concentration of type I IFN used (26, 27, 28, 29, 30). Recently, Biron (25) proposed a model in which the level of type I IFN induced by a pathogen determines the production of IL-12 and the type of immune responses. According to this model, one would assume that ODN 2216 as a potent inductor of IFN- in plasmacytoid DC (13) is able to support a CD8 recall response and to activate NK cells, but would not promote priming of a CD8 T cell response. Indeed, our recent results demonstrate that CpG-A (ODN 2216) does not promote priming of melan A peptide-specific CD8 T cells in PBMC. In contrast, CpG-B, which supports IL-12 production in plasmacytoid DC, but is poor at inducing type I IFN (ODN 2006) (12), enhanced priming of CD8 T cells.5
In conclusion, our results revealed that soluble factors induced by CpG-A within PBMC drive monocytes toward a DC-like phenotype that preferentially promotes the expansion of memory CD8 T cells. It has to be considered that the results presented were obtained from circulating cell populations that might differ from the immunologic events in tissues. Furthermore, the CpG-A-induced DC type may be of limited value for ex vivo vaccination strategies, but it may improve our understanding of the immune responses elicited by CpG-A administered in vivo, and will help to appropriately design clinical studies testing CpG-A for the therapy of viral disease and cancer.
|
Footnotes |
|---|
2 A.K. and S.R. contributed equally to this study.
3 Address correspondence and reprint requests to Dr. Gunther Hartmann, Abteilung für Klinische Pharmakologie, Medizinische Klinik Innenstadt, Ziemssenstrasse 1, 80336 Munich, Germany. E-mail address: ghartmann{at}lrz.uni-muenchen.de
4 Abbreviations used in this paper: ODN, oligodeoxynucleotides; CD40L, CD40 ligand; CpG-CM, CpG-conditioned medium; DC, dendritic cell; FSC, forward light scatter; MFI, mean fluorescence intensity; PDC, plasmacytoid DC; SSC, side light scatter; TLR, Toll-like receptor.
5 S. Rothenfusser, V. Hornung, M. Ayyoub, S. Britsch, A. Towarowski, A. Krug, N. Lubenow, D. Speiser, S. Endres, and G. Hartmann. Two distinct types of CpG oligonucleotides differentially enhance human peptide-specific CD8+ T cell responses in vitro. Submitted for publication.
Received for publication July 8, 2002. Accepted for publication January 15, 2003.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
/ in plasmacytoid dendritic cells. Eur. J. Immunol. 31:2154.[Medline]
. J. Exp. Med. 179:1109. and as immune regulators: a new look. Immunity 14:661.[Medline]
and granulocyte-macrophage colony-stimulating factor differentiate peripheral blood monocytes into potent antigen-presenting cells. J. Leukocyte Biol. 64:358.[Abstract]
induce CD86 (B7.2) expression and accelerate dendritic cell maturation from human peripheral blood mononuclear cells. Scand. J. Immunol. 50:499.[Medline]
T cells via interferon-/-. Eur. J. Immunol. 31:3525.[Medline]
/ inhibition of interleukin 12 and interferon- production in vitro and endogenously during viral infection. Proc. Natl. Acad. Sci. USA 94:634. interferes with the differentiation of dendritic cells from peripheral blood mononuclear cells: selective inhibition of CD40-dependent interleukin-12 secretion. J. Interferon Cytokine Res. 19:471.[Medline]
and - inhibit the in vitro differentiation of immunocompetent human dendritic cells from CD14+ precursors. Blood 96:210.This article has been cited by other articles:
|
|
 |
|
  M. L. Salem, C. M. Diaz-Montero, A. A. Al-Khami, S. A. El-Naggar, O. Naga, A. J. Montero, A. Khafagy, and D. J. Cole Recovery from Cyclophosphamide-Induced Lymphopenia Results in Expansion of Immature Dendritic Cells Which Can Mediate Enhanced Prime-Boost Vaccination Antitumor Responses In Vivo When Stimulated with the TLR3 Agonist Poly(I:C) J. Immunol., February 15, 2009; 182(4): 2030 - 2040. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Merino, S. Nogueras, T. Garcia-Maceira, M. Rodriguez, A. Martin-Malo, R. Ramirez, J. Carracedo, and P. Aljama Bacterial DNA and endothelial damage in haemodialysis patients Nephrol. Dial. Transplant., November 1, 2008; 23(11): 3635 - 3642. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 U. Bhan, G. Trujillo, K. Lyn-Kew, M. W. Newstead, X. Zeng, C. M. Hogaboam, A. M. Krieg, and T. J. Standiford Toll-Like Receptor 9 Regulates the Lung Macrophage Phenotype and Host Immunity in Murine Pneumonia Caused by Legionella pneumophila Infect. Immun., July 1, 2008; 76(7): 2895 - 2904. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. O. Kvale, J. Dalgaard, F. Lund-Johansen, H. Rollag, L. Farkas, K. Midtvedt, F. L. Jahnsen, J. E. Brinchmann, and J. Olweus CD11c+ dendritic cells and plasmacytoid DCs are activated by human cytomegalovirus and retain efficient T cell-stimulatory capability upon infection Blood, March 1, 2006; 107(5): 2022 - 2029. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Jiang, M. M. Lederman, J. R. Salkowitz, B. Rodriguez, C. V. Harding, and S. F. Sieg Impaired Monocyte Maturation in Response to CpG Oligodeoxynucleotide Is Related to Viral RNA Levels in Human Immunodeficiency Virus Disease and Is at Least Partially Mediated by Deficiencies in Alpha/Beta Interferon Responsiveness and Production J. Virol., April 1, 2005; 79(7): 4109 - 4119. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. U. Saikh, T. L. Kissner, A. Sultana, G. Ruthel, and R. G. Ulrich Human Monocytes Infected with Yersinia pestis Express Cell Surface TLR9 and Differentiate into Dendritic Cells J. Immunol., December 15, 2004; 173(12): 7426 - 7434. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Wysocka, B. M. Benoit, S. Newton, L. Azzoni, L. J. Montaner, and A. H. Rook Enhancement of the host immune responses in cutaneous T-cell lymphoma by CpG oligodeoxynucleotides and IL-15 Blood, December 15, 2004; 104(13): 4142 - 4149. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Vollmer, M. Jurk, U. Samulowitz, G. Lipford, A. Forsbach, M. Wullner, S. Tluk, H. Hartmann, A. Kritzler, C. Muller, et al. CpG oligodeoxynucleotides stimulate IFN-{gamma}-inducible protein-10 production in human B cells Innate Immunity, December 1, 2004; 10(6): 431 - 438. [Abstract] [PDF]
|
|
|
|
|
|
J. C. Deng, T. A. Moore, M. W. Newstead, X. Zeng, A. M. Krieg, and T. J. Standiford CpG Oligodeoxynucleotides Stimulate Protective Innate Immunity against Pulmonary Klebsiella Infection J. Immunol., October 15, 2004; 173(8): 5148 - 5155. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Vollmer, R. D. Weeratna, M. Jurk, H. L. Davis, C. Schetter, M. Wullner, T. Wader, M. Liu, A. Kritzler, and A. M. Krieg Impact of modifications of heterocyclic bases in CpG dinucleotides on their immune-modulatory activity J. Leukoc. Biol., September 1, 2004; 76(3): 585 - 593. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Rothenfusser, V. Hornung, M. Ayyoub, S. Britsch, A. Towarowski, A. Krug, A. Sarris, N. Lubenow, D. Speiser, S. Endres, et al. CpG-A and CpG-B oligonucleotides differentially enhance human peptide-specific primary and memory CD8+ T-cell responses in vitro Blood, March 15, 2004; 103(6): 2162 - 2169. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Wagner, H. Poeck, B. Jahrsdoerfer, S. Rothenfusser, D. Prell, B. Bohle, E. Tuma, T. Giese, J. W. Ellwart, S. Endres, et al. IL-12p70-Dependent Th1 Induction by Human B Cells Requires Combined Activation with CD40 Ligand and CpG DNA J. Immunol., January 15, 2004; 172(2): 954 - 963. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
