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CUTTING EDGE |
* Immunology Division and the Cooperative Research Centre for Vaccine Technology, Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia; Now at Bavarian Nordic, Munich, Germany; and Department of Immunology, Peking University Health Science Center, Beijing, China
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Abstract |
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; 2) developmental dependence on, and mRNA expression of, IFN regulatory factor-8; 3) mRNA expression of TLRs and chemokine receptors; 4) production of IL-12 p40/70, IFN-, MIP-1, and RANTES in response to TLR ligands; 5) expression of cystatin C; and 6) cross-presentation of exogenous Ag to CD8 T cells. Furthermore, despite lacking surface CD8 expression, the CD24high subset contained CD8 mRNA and up-regulated surface expression when transferred into mice. This culture system allows access to bona fide counterparts of the splenic DC subsets. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionDisclosuresReferences |
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There are several well-established procedures for generating DC in culture from bone marrow (BM) precursors or from blood monocytes using GM-CSF and IL-4 (GM/IL-4 DC) (6). However, GM/IL-4 DC do not seem to show the heterogeneity in DC phenotype and function found with splenic DC. In fact, it is not clear whether GM/IL-4 DC have any counterparts among steady-state DC in vivo. In relation to this, a recent study found the development of steady-state DC was Stat3 dependent, whereas GM/IL-4 DC development from monocytes was Stat3 independent, suggesting that these two groups of DC have distinct developmental origins (7).
Culture of BM with fms-like tyrosine kinase 3 (flt3) ligand (FL) is a more recent method that allows the generation of both cDC and pDC in large numbers (now referred to as FL-DC) (8, 9, 10). The dependency on FL for DC generation in this culture system correlates with the observations that FL–/– mice have reduced DC numbers (11), that injection of mice with FL greatly increases DC numbers (12, 13), and that only early BM progenitors that express flt3 are effective precursors of DC (14). Some initial observations suggested that FL-cDC may further divide into subsets, but their relationship to splenic DC subsets was not further investigated (10, 15).
In this study, we show that despite the presence of some CD11b on all cDC from FL cultures, and the absence of surface CD4 and CD8 expression, FL-DC could be clearly segregated by surface markers into the equivalents of steady-state splenic pDC, CD8+ cDC, and CD8– cDC. The shared properties between the FL-DC subsets and the splenic DC subset counterparts included surface marker expression, transcription factor expression and dependence for development, ability to cross-present cellular Ag to CD8 T cells, expression of TLR and chemokine receptors, and production of cytokines and chemokines in response to TLR stimulation. This system should allow access to large numbers of the DC subsets for further study. In particular, up to 25 x 106 of the CD8+ cDC equivalents can be generated from culturing the BM of one mouse with FL.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionDisclosuresReferences |
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C57BL/6J (Ly5.2), C57BL/6 Pep3b CD45.1 (Ly5.1) OT-I, OT-II, bm1, and IRF-8–/– mice (from I. Horak (Free University of Berlin, Berlin, Germany) and A. Kallies (Walter and Eliza Hall Institute)) were bred under specific pathogen-free conditions and used at 7–10 wk of age.
FL-DC preparation
FL-DC were generated as described (8, 10, 16), with some modifications. BM was extracted, and red cells were removed by a 30-s exposure to 0.168 M NH4Cl and washed three times. Cells were cultured at 1.5–3 x 106 cells/ml in modified mouse osmolarity RPMI 1640 culture medium (DC medium) (17) containing 300 ng/ml murine FL (generated in-house), for 8–10 days at 37°C in 10% CO2. Four-color staining with mAbs to CD11c, CD45RA, CD24, and CD11b was used to separate the DC subsets.
Splenic DC preparation
Recovery of splenic DC was performed as previously described (2, 5). Briefly, spleens were digested with DNase and collagenase, and light-density cells were collected after centrifugation in 1.077 g/cm3 Nycodenz. Cells were incubated with rat mAbs against mouse CD19 (ID3), CD3 (KT31.1), Thy1.1 (T24/31.7), TER-119, and Ly6G (1A8), and non-DC depleted using anti-rat Ig magnetic beads (Qiagen).
Flow cytometry
DC were stained with varying combinations of mAbs to CD11c (N418-A594 or allophycocyanin), CD45RA (14.8-PE), CD11b (M1/70-Cy5), CD24 (M1/69-FITC or biotin), CD4 (GK1.5-allophycocyanin), CD8 (YTS169.4-FITC, PE, or allophycocyanin), signal regulatory protein (SIRP)- (p84-biotin) and Ly5.2 (S450-15.2-FITC), with second-stage staining with streptavidin-PerCP-Cy5.5 or streptavidin-PE. Cell sorting and analysis was performed on a FACSVantageSE DiVa, FACStarPlus, LSR (BD Biosciences), or MoFlo (DakoCytomation) instruments.
In vivo FL-DC transfers
Between 0.4 and 2 x 106 sorted FL-DC subsets from Ly5.2 BM cultures were injected i.v. into nonirradiated Ly5.1 mice. Three days later, spleens were harvested and enriched for DC before flow cytometric analysis.
Ag presentation assays
OVA-specific CD8 (OT-I) or CD4 (OT-II) T cells were obtained from lymph nodes by depletion of non-CD8 or non-CD4 T cells, respectively, and CFSE labeled, as described elsewhere (18, 19). For T cell activation, 5 x 103 sorted DC were incubated with a titration of either SIINFEKL (MHC class I (MHC I)) peptide for 45 min at 37°C and then washed three times, or a titration of OVA323–339 (MHC class II (MHC II)) peptide (Auspep) present during the duration of the experiment. For cross-presentation assays, 2.5 x 104 sorted DC were cultured with varying numbers of gamma-irradiated bm1 splenocytes that were previously coated with 10 mg/ml OVA protein and washed twice. For all cultures, 5 x 104 T cells were added and cultured in DC medium for 60 h with 10 ng/ml GM-CSF (PeproTech). At this time, cells were stained for V2-PE and CD8-allophycocyanin or CD4-allophycocyanin, and PE+allophycocyanin+CFSElow cells were quantitated by flow cytometry.
Western blot analysis
Western blot of cell lysates was performed as described (20). Actin and cystatin C (CyC) were detected using anti-actin (Sigma-Aldrich) and anti-human CyC (DakoCytomation) rabbit sera.
RT-PCR
RT-PCR was performed on the purified DC subsets using primers for IFN regulatory factor (IRF)-4, IRF-8, CD4, and CD8. Real-time PCR for TLRs and chemokine receptors was performed using the QuantiTect SYBR Green PCR kit (Qiagen). Primer sequences and PCR conditions are available on demand.
DC activation
Sorted DC (1 x 105) were cultured in duplicate, either in 200 µl of DC medium alone or with 1 µg/ml R848 (Invitrogen Life Technologies), 1 µM CpG 2216 (Geneworks), or 1 µg/ml LPS (Sigma-Aldrich) for 24 h. For IL-12 p40/p70 and IFN- production, DC were cultured in DC medium with 100 ng/ml GM-CSF, 20 ng/ml IL-4, and 20 ng/ml IFN-
, with or without 0.5 µM CpG 2216, for 48 h.
ELISA
ELISAs were performed as previously described (21) with capture mAbs to IL-12 p40/p70 (C16.6 or R29A5), IL-6 (MP5-20F3), RANTES (53433), MIP1 (39624.11), or IFN- (RMMA-1), and detection via biotinylated mAbs to IL12 p40/p70 (C17.8), IL-6 (M35-32C11), RANTES (R&D Systems), MIP-1 (R&D Systems), or polyclonal rabbit Ab to IFN- (PBL).
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionDisclosuresReferences |
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A total of 
5 x 106 DC is usually isolated from one mouse spleen, whereas 50 x 106 DC could be generated from culturing the BM of one mouse with FL (Fig. 1A). To assess phenotype, DC derived from FL-stimulated BM cultures were compared in surface Ag expression to freshly isolated splenic DC. Both FL culture-derived and splenic CD11c+ DC contained distinct CD45RA– cDC and CD45RA+ pDC populations (Fig. 1A), as others have noted previously (8, 9, 16). However, because the cDC component had been shown to express both surface CD11b and CD205 (8), but not CD4 or CD8, the markers normally used to segregate the splenic CD8+ and CD8– cDC subtypes were not applicable for FL-cDC subset discrimination (1, 17) (Fig. 1).
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, recently identified by microarrays as being selectively expressed by CD8– cDC (M. Lahoud, A. Proietto, and K. Shortman, manuscript in preparation) and differentially expressed among rat DC (23). FL-cDC expressed these markers, as well as CD11b, at different levels, which allowed their further division into two subtypes: CD24highSIRP-lowCD11blow and CD24lowSIRP-highCD11bhigh (Fig. 1A, top panels). We found that four-color staining for CD11c, CD45RA, CD11b, and CD24 was the ideal combination for discrimination of the three FL-DC subtypes. An almost identical separation of subsets using the same combination of markers was found with splenic DC, but with differences in the relative proportions of each (Fig. 1A, lower panels). In particular, there was a larger proportion of CD24high cells among the FL-DC, the putative CD8+ cDC equivalent. Also, whereas the splenic cDC subsets are selective in their surface expression of CD11b and CD205 in the steady state (5), all FL-cDC expressed some levels of each (Fig. 1A) (8, 9). This is not surprising considering that all splenic cDC up-regulate CD11b and CD205 upon in vitro culture (17). In view of these similarities in aspects of surface phenotype, we tested whether the FL-DC and splenic DC populations were developmentally and functionally equivalent. The terminology used hereafter for the three subsets is pDC, CD24high DC (CD8+ cDC equivalents), and CD11bhigh DC (CD8– cDC equivalents).
DC subset differences in IRF-8–/– mice
IRF-8 transcription factor knockout mice retain splenic CD8– cDC but have reduced numbers of pDC and CD8+ cDC (Fig. 1B, lower panel) (24). To assess whether a related defect occurred within FL-DC, we cultured BM from IRF-8–/– and wild-type (WT) mice with FL and compared the DC produced. Among the IRF8–/– FL-DC, there was comparable production of CD11bhigh DC but greatly reduced numbers of pDC and CD24high DC compared with WT FL-DC (Fig. 1B, top panel, and our unpublished data). Interestingly, a converse deficit in DC subset production has been reported using IRF-4–/– mice, which have an absence of CD4+ DC in vivo and an absence of CD11bhigh DC in FL BM cultures (15).
IRF expression
Not only do the different spleen DC subsets require particular IRFs for their normal development, they also vary in their expression of these transcription factors in the steady state: the CD8+ DC mainly express IRF-8, CD8– DC mainly express IRF-4, whereas pDC express both (15, 25). The FL-DC subsets were examined for IRF-4 and IRF-8 mRNA. Indeed, the expression pattern of IRFs in FL-DC subsets correlated with those in splenic DC (Fig. 2A), in line with previous studies (15).
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Even though unstimulated FL-DC do not express surface CD4 or CD8, unlike their putative in vivo counterparts, mRNA expression of these molecules was tested. CD24high DC and pDC did express some CD8 transcript, whereas CD11bhigh DC did not. Only pDC expressed CD4 transcript (Fig. 2A). To see whether surface expression might be dependent on the normal in vivo DC environment, we transferred each purified FL-DC subset in vivo. After 3 days, we compared host and donor-derived splenic DC for surface CD4 and CD8 expression (Fig. 2B). Almost 75% of the recovered CD24high DC had up-regulated CD8, but very few expressed CD4. Transferred FL-pDC up-regulated both CD4 and CD8, so they then resembled their in vivo counterparts (2). However, transferred CD11bhigh DC up-regulated very little surface CD4 or CD8 in this time (15).
Ag presentation and cross-presentation
To assess the capacity of the cDC subsets to activate naive T cells, DC were coated with synthetic OVA peptides for MHC I or II, and then incubated with OT-I CD8 T cells or OT-II CD4 T cells, respectively. FL-DC showed comparable efficiency to their splenic DC counterparts in stimulating the proliferation of naive CD8 (Fig. 3A) and CD4 T cells (B). Cross-presentation, a process whereby exogenous Ags are taken up and presented via MHC I, is conducted efficiently by splenic CD8+ cDC but not CD8– DC nor pDC (19, 26). To examine whether any of the FL-cDC populations were able to cross-present, we incubated each subset with OT-I cells and cellular Ag in the form of OVA protein-coated gamma-irradiated splenocytes from bm1 mice (unable to present OVA to OT-I cells). Of the FL-DC, only the CD24high subset was able to cross-present with efficiency similar to splenic CD8+ cDC (Fig. 3C).
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In the steady state, the splenic CD8+ cDC are unique among the DC subsets in their expression of the cysteine protease inhibitor CyC (20), although the precise role of CyC in CD8+ cDC biology is still controversial (20, 27). We found that only CD24high FL-DC expressed significant levels of CyC (Fig. 3D).
TLR expression
The splenic DC subsets have unique TLR expression patterns, which enable them to directly respond to particular TLR ligands (28, 29). Splenic pDC express TLR7 and -9 but not TLR3 or -4; CD8+ cDC express TLR3, -4, and -9 but not TLR7; CD8– cDC express TLR4, -7, and -9, but not TLR3. Using real-time PCR, an almost identical pattern of TLR mRNA expression was found between the FL-DC and splenic DC subset equivalents (Fig. 4A).
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The spleen DC subsets show quantitative differences in chemokine receptor expression, with pDC being the highest expressors of CXCR3 and CCR9, and CD8– cDC the highest expressors of CCR6 and CX3CR1, whereas CD8+ cDC express some CXCR3 and CX3CR1 (29). We found comparable mRNA expression of most of these receptors between the FL-DC and splenic DC (Fig. 4B). One striking difference was the lack of CCR6 expression in CD11bhigh FL-DC compared with splenic CD8– cDC, which suggests an in vivo factor may be required for its normal expression.
Cytokine and chemokine production
A major functional difference between the splenic DC subsets is their capacity to produce particular cytokines. Cytokine production in response to TLR engagement depends on the TLR expression pattern of the DC subset, the TLR ligand, and on other factors such as the cytokine environment. Moreover, the splenic DC subsets differ in the cytokines they produce to the same TLR ligand (21, 29). For example, only pDC and CD8– cDC express TLR7 and produce cytokines in response to the TLR7 ligand, R848. In contrast, all splenic DC express TLR9, but when stimulated with the TLR9 ligand, CpG DNA, pDC produce the highest levels of IFN-, whereas CD8+ cDC produce the highest levels of IL-12 p70. FL-DC subsets were compared with their splenic DC counterparts for cytokine production, in response to R848, CpG 2216, or LPS, in conditions known to induce cytokine production, and the supernatants were analyzed by ELISA. The patterns of cytokine production by the FL-DC subsets reflected their splenic DC equivalents (Fig. 5A).
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. The CD24high DC subsets showed negligible chemokine or IL production in response to R848, correlating to their lack of TLR7 expression. Conclusions
We have shown that FL BM cultures produce DC subsets that, despite differences in CD4 and CD8 expression, are close equivalents of the steady-state splenic pDC, CD8+ cDC, and CD8– cDC subtypes. Importantly, key functional differences are conserved between the FL-DC and spleen DC equivalents. The major novel finding in this study is the generation of a functionally distinct spleen CD8+ cDC equivalent, and a strategy for its isolation in high yield. We thus propose that, in contrast to GM/IL-4 DC, FL cultures better facilitate the study of steady-state DC subset ontogeny and function. Finally, FL-stimulated cultures may be useful for identifying and producing human DC counterparts to the murine DC subsets.
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Acknowledgments |
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Disclosures |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionDisclosuresReferences |
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Footnotes |
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1 This work was supported by the Australian Government’s Cooperative Research Centres (CRC) Program and the National Health and Medical Research Council. S.H.N. is supported by scholarships from the University of Melbourne and the CRC for Vaccine Technology, and A.I.P. is supported by an Australian Postgraduate Award. 
2 S.H.N. and A.I.P. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Shalin H. Naik or Dr. Li Wu, Walter and Eliza Hall Institute, 1G Royal Parade, Parkville, Victoria, Australia. E-mail addresses: naik{at}wehi.edu.au and wu{at}wehi.edu.au
4 Abbreviations used in this paper: DC, dendritic cell; pDC, plasmacytoid pre-DC; cDC, conventional DC; BM, bone marrow; GM/IL-4 DC, GM-CSF and IL-4 DC; FL, fms-like tyrosine kinase 3 (flt3) ligand; MHC I, MHC class I; MHC II, MHC class II; IRF, IFN regulatory factor; CyC, cystatin C; SIRP, signal regulatory protein; WT, wild type.
Received for publication March 15, 2005. Accepted for publication April 12, 2005.
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionDisclosuresReferences |
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+ mouse spleen dendritic cells do not originate from the CD8– dendritic cell subset. Blood 102: 601-604.. J. Exp. Med. 179: 1109-1118.– dendritic cell development. Proc. Natl. Acad. Sci. USA 101: 8981-8986. production via Toll-like receptor 9-dependent and -independent pathways. Proc. Natl. Acad. Sci. USA 101: 11416-11421., and IFN- by mouse dendritic cell subsets. J. Immunol. 166: 5448-5455.+ dendritic cells. J. Exp. Med. 196: 1415-1425.+ DC correlates with unresponsiveness to imidazoquinolines. Eur. J. Immunol. 33: 827-833.[Medline]
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