SEARCH:   Advanced

Prepublished online as a Blood First Edition Paper on March 20, 2003; DOI 10.1182/blood-2002-10-3186. This Article Abstract Full Text (PDF) All Versions of this Article: 2002-10-3186v1 102/2/601    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Naik, S. Articles by Shortman, K. Search for Related Content PubMed PubMed Citation Articles by Naik, S. Articles by Shortman, K. Related Collections Immunobiology Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 15 July 2003, Vol. 102, No. 2, pp. 601-604 IMMUNOBIOLOGY CD8+ mouse spleen dendritic cells do not originate from the CD8- dendritic cell subset Shalin Naik, David Vremec, Li Wu, Meredith O'Keeffe, and Ken Shortman From The Walter and Eliza Hall Institute of Medical Research and the Cooperative Research Centre for Vaccine Technology, Melbourne, Victoria, Australia    Abstract Top Abstract Introduction Materials and methods Results Discussion References   Although previous studies had indicated that the CD8- and CD8+ subtypes of murine dendritic cells (DCs) differ in immediate origin, a recent study found that intravenous transfer of CD8- DCs led to CD8+ DCs in the spleen several days later, suggesting a direct precursor-product relationship. We have repeated these experiments with a balance sheet approach. We find that though a few CD8+ DCs can be generated in such experiments, this is a rare event and could be the result of a contaminant precursor. Most of the immediate precursors of CD8+ DCs are cells that lack the phenotype of a recognizable DC. CD8- DCs and CD8+ DCs are not precursor-product related, though these sublineages may be connected further upstream.    Introduction Top Abstract Introduction Materials and methods Results Discussion References   The CD8+ and CD8- subsets of dendritic cells (DCs) from mouse spleen are of special interest because, as freshly isolated cells, they differ in functional characteristics.1 However, the developmental origin of these DC subtypes and their relationship to each other has been controversial. The original view that splenic CD8+ DCs were of lymphoid origin and that splenic CD8- DCs were of myeloid origin has been shown to be incorrect.2,3 Differences in the cytokine and transcription factor requirements suggest that the developmental pathways involved differ.4-8 Culture of purified splenic CD8+ DCs or splenic CD8- DCs has failed to show any transformation from one to the other,9 though evidence indicates that CD8 can be induced to at least moderate levels on some DC subtypes.10,11 An in vivo kinetic study, using continuous 5-bromodeoxyuridine (BrdU) labeling, showed no sign of a precursor-product relationship between CD8- and CD8+ DCs.9 In direct conflict with this is the conclusion in a recent paper by Martinez del Hoyo et al.12 They isolated CD8- DCs, transferred them intravenously into a Ly5-disparate, nonirradiated recipient, and found 3 to 4 days later CD8+ DCs of donor origin. They concluded that CD8+ DCs normally originate from CD8- DCs—the 2 are merely maturation stages of the same lineage. In view of the conflict, we have now carried out similar experiments. Although we did obtain some CD8+ DCs on transfer of a CD8- DC preparation, recoveries were extremely low, and we argue this is an uncommon event that might be attributed to a contaminant precursor. We find that most of the immediate precursors of CD8+ DCs are cells lacking normal DC characteristics.    Materials and methods Top Abstract Introduction Materials and methods Results Discussion References   Mice C57BL/6J WEHI (Ly5.2) mice 6 to 8 weeks of age were used as donors, and C57BL/6 Pep3b mice (Ly5.1) of a similar age range were used as nonirradiated recipients. Preparation of CD8- DCs The method used, described previously,13 was similar to that of Martinez del Hoyo et al,12 except that fluorescence-activated cell sorting (FACS) rather than immunomagnetic microbead selection was used in the final separation. Samples of the original spleen suspension (after red cell and dead cell removal14) and of cells at all stages of the procedure were retained for counting and assay of precursor activity. Briefly, spleens were digested for 20 minutes at room temperature with collagenase-DNAase then were treated with ethylenediaminetetraacetic acid (EDTA). All subsequent procedures were performed at 0°C to 4°C in an EDTA-salt solution. Light-density cells, 5% of the total nucleated spleen cells, were selected by centrifugation in a 1.077 g/cm3 Nycodenz medium. Non-DC and plasmacytoid cells were depleted by incubation with rat monoclonal antibody (mAb) to CD3, CD45R (B220), Thy-1, Gr-1, and erythrocytes (TER-119), and they were removed using antirat immunoglobulin immunomagnetic beads. Autofluorescent cells were removed by rapid presorting of the unlabeled preparation or by gating out at the time of sorting.13 Control experiments indicated an average 50%, and never more than 70%, of DCs were lost during these enrichment procedures. The resultant DC-enriched preparation, approximately 2% of the original spleen, was usually stained for CD11c (N418-fluorescein), major histocompatibility complex 2 (MHC 2) (M5/114-Alexa 594), and CD8 (YTS169.4-Cy5) and for the CD11chiMHC 2hiCD8- fraction selected. Alternatively, the DC-enriched preparation, 50% to 60% pure and representing approximately 2% of the original spleen, was usually stained for one of these markers, and cells with negative, medium, and high fluorescence above background were sorted. Reanalysis of the CD11chiMHC 2hiCD8- fraction gave a purity of 99%, with no CD8+ DCs detected. Assay for immediate precursors of CD8+ DCs The assay used was based on that used by Martinez del Hoyo et al.12 Samples of various DC-enrichment fractions were transferred intravenously (1-10 x 106 cells per mouse) into 2 or more nonirradiated Ly5.1 recipients. Three days later, the spleens of recipient mice were pooled. DCs were then extracted and enriched by density separation and immunomagnetic bead depletion, as described,13 to allow clean analysis of donor-derived DCs. Cells were counted and stained for donor-type Ly5.2 (ALI-4A2-biotin with streptavidin-phycoerythrin or streptavidin-Alexa 594 second stage), for CD11c (N418-fluorescein), for CD8 (YTS169.4-Cy5), and in some experiments for CD205 (NLDC-145). The number of donor-derived CD8+ DCs produced per cell transferred was then determined as a measure of precursor activity.    Results Top Abstract Introduction Materials and methods Results Discussion References   Generation of CD8+ DCs from the CD11chiMHC 2hiCD8- DC fraction We first attempted to repeat the experiment whereby the transfer of purified CD8- splenic DCs produced CD8+ DCs.12 However, to obtain high purity and to segregate different levels of marker expression, we used FACS rather than immunomagnetic particle separation in the final separation of CD8- DCs. We additionally selected cells bearing relatively high levels of MHC 2, therefore defining more tightly the DC population. Purity was close to 99% on reanalysis, so approximately 1% of the preparation represented possible contaminants. These cells (3-5 x 106) were then transferred intravenously into nonirradiated Ly5.1 recipients. A DC-enriched preparation was made 3.5 days later from the pooled recipient spleens, and the donor-derived (Ly5.2+) cells were gated and analyzed. In 3 of 7 transfer experiments, donor-derived DCs were detected in the recipient spleens. From 5% to 30% of these donor-derived DCs were CD8+ (Figure 1) and DEC-205+, whereas the rest remained CD8- or stained at only low levels for CD8. Similar results were obtained at days 2 and 4. Thus, we sometimes did see the generation of CD8+ DCs from a CD8- DC fraction, though, in contrast to the previous study,12 only a minority showed the acquisition of CD8. Given that most splenic DCs turn over in 3 days,9 most donor-derived DCs should have been CD8+ if they originated from the CD8- DC subset. View larger version (14K): [in this window] [in a new window]   Figure 1.. Characterization of donor-derived DCs after transfer of purified CD8- DCs. Splenic CD8-CD11chiMHC 2hi DCs were isolated and sorted from Ly5.2 mice and transferred intravenously into nonirradiated Ly5.1 recipients (5 x 106 per mouse). After 3.5 days, DCs were isolated from the pooled recipient spleens, and Ly5.2+CD11c+ DCs were analyzed for CD8 expression. Histograms indicate Ly5.2 gated cells either unstained (gray line) or stained (black line) for CD8, with the vertical line indicating the cutoff for levels of CD8 on CD8hi DCs. Of 7 transfer experiments, 4 gave no detectable donor-derived DCs, whereas 3 gave donor-derived DCs containing 5% to 33% CD8+ DCs. The experiment presented is the one showing the highest donor cell recoveries and the highest proportion of Ly5.2+CD8+ DCs.   Recovery of DCs on transfer of the CD8- DC fraction In addition to the low proportion of DCs that became CD8+, a disturbing aspect of the experiment was the low overall recovery rate of the transferred DCs in the spleens of the recipient mice (Table 1). This was true at all time points studied. The highest recovery rate measured in these experiments was 0.33% of the CD8- DCs transferred, of which only approximately one quarter were CD8+ DCs (Table 1). Even allowing for a 50% loss of DCs in the enrichment procedure preceding analysis, at most 0.16% of the transferred DCs produced CD8+ DCs in the recipient spleens. View this table: [in this window] [in a new window]   Table 1.. Recovery of transferred cells   This is in line with the reported values of Martinez del Hoyo et al,12 from which it can be calculated that only 0.3% of the transferred cells were recovered as CD8+ DCs. Because this is within the contaminant level of the transferred CD8- DCs, it was not proven that it was the CD8- DCs themselves that produced the CD8+ DCs. Distribution of the immediate precursors of CD8+ DCs during DC enrichment It could still be argued that this observed production of CD8+ DCs, though derived from only 0.3% of the CD8- DC fraction injected, was representative of the other 99.7% transferred but lost. If all the immediate precursors of CD8+ DCs were indeed CD8- DCs, such precursor activity, as measured by the ability to produce CD8+ DCs in the spleen 3 days after transfer, should separate with CD8- DCs and therefore be more than 100-fold enriched in a pure CD8- DC fraction. The distribution of such immediate CD8+ DC precursor activity in fractions sampled during the steps of DC isolation was therefore determined after intravenous transfer of a counted number of cells. Precursor activity was determined as specific activity (donor-derived CD8+ DCs per recipient spleen per 106 donor cells transferred) and then calculated as total activity (specific activity x 10-6 multiplied by the number of cells in each separation fraction from one donor spleen) (Table 2). Specific activity allows a measure of precursor enrichment, and total activity allows a measure of precursor recovery. View this table: [in this window] [in a new window]   Table 2.. Immediate precursors of CD8+ DCs are not all recovered in the DC-enriched fractions   To gauge the enrichment and recovery of CD8+ DC precursors concomitant with CD8- DC enrichment, the total CD8+ DC precursor potential of a spleen was first determined. A suspension of spleen cells, with dead cells and erythrocytes removed, was counted and injected intravenously, and the number of donor-type CD8+ DCs arising after 3.5 days was determined. We calculated the per-cell–specific precursor activity and then multiplied this by the number of viable nucleated cells obtained from each donor spleen to give an initial total precursor activity figure for balance sheet purposes (Table 2). The first step in DC purification involved separating a light-density (less than 1.077 g/cm3) fraction, which included only approximately 5% of the viable nucleated spleen cells but virtually all of the fully developed DCs. Although this fraction gave some enrichment of CD8+ DC precursor–specific activity (approximately 5-fold), it was less than the 20-fold obtained for DCs. More important, the balance sheet of CD8+ DC total precursor activity showed that 70% was eliminated with the dense cells (Table 2). Additional studies showed that most of the immediate precursors of CD8+ DCs were within the 28% of spleen viable nucleated cells, with density ranging between 1.083g/cm3 and 1.077 g/cm3. Further enrichment of fully developed DCs, by successive immunomagnetic bead depletion of non-DC lineage cells and sorting to remove autofluorescent macrophages, enriched/DC purity to 70% to 80% but gave little further enrichment of CD8+ DC precursor–specific activity (Table 2). The final DC-enriched fraction was only 5-fold enriched in specific CD8+ precursor activity, and it represented only 11% of the total precursor activity in the original spleen suspension. Much of this apparent activity could have resulted from the seeding and persistence of preformed CD8+ DCs rather than their generation from precursors. Thus, most of the splenic CD8+ DC precursors were not CD8- DCs. Distribution of CD8+ DC precursor activity during sorting for CD8- DCs Because some apparent CD8+ DC precursor activity did persist in the DC-enriched fraction, we asked whether CD8- DCs could be a real, if minor, source of CD8+ DCs. The enriched fraction (70%-80% DCs) was therefore segregated according to surface expression of the 3 markers used to define CD8- DCs—CD11c, MHC 2, and CD8. Labeling the DCs with antibodies to these markers did not result in any loss of CD8+ DC precursor activity (Table 2). On sorting the labeled cells, precursor activity was found distributed over many fractions (Table 3), not just in the CD8-, CD11chi, or MHC 2hi fractions. On separation by CD8 expression levels, some "activity" was associated with the CD8 high fraction, and this presumably represented the seeding and persistence of pre-existing CD8+ DCs. Even more of the total and specific activity was found in the CD8 intermediate fraction, however, suggesting the presence of less mature cells en route to producing CD8 high DCs (Table 3). On separation according to CD11c or MHC 2 expression, the greatest enrichment of activity was in the fractions expressing levels of CD11c and MHC 2 that were lower than those of fully developed DCs and that might therefore contain early DC forms (Table 3). In no case was the precursor activity enriched along with the markers of CD8- DCs (MHC 2hi, CD11chi, or CD8-), though some measurable activity was usually associated with these markers, as in Figure 1. When all 3 markers were used together to isolate CD8-CD11chiMHC 2hi DCs (as in Figure 1), a mean of 1.1% of the total initial spleen precursor activity was recovered in the 3 of 7 experiments when any CD8+ DCs were found in the recipient spleens (data not shown). View this table: [in this window] [in a new window]   Table 3.. Immediate precursors of CD8+ DCs in DC-enriched spleen preparations do not segregate with the markers of mature CD8- DCs      Discussion Top Abstract Introduction Materials and methods Results Discussion References   In at least some of our experiments, CD8+CD205+ splenic DCs were generated when purified samples of CD8- DCs were transferred intravenously, in accordance with the observations of Martinez del Hoyo et al.12 However, our conclusions differ radically from theirs because we demonstrate that most immediate precursors of CD8+ DCs are not CD8- DCs at all but are cells lacking the characteristics of mature DCs and so are lost on DC enrichment. Our results highlight the importance of a balance sheet approach, examining activity at all enrichment steps rather than only determining the activity of an enriched candidate cell. A major limitation of the earlier study was the extremely low recovery of DCs in the spleen following intravenous transfer. We also found this with at most only 0.16% of the sorted CD8- DCs transferred, forming CD8+ DCs in the recipient spleens. This poor recovery of DCs after intravenous transfer is compounded by the separate problem of poor recovery of CD8- DC precursor activity during DC enrichment and purification. When we did recover any CD8+ DC precursor activity in a highly purified, sorted CD8-CD11chiMHC 2hi fraction, it represented only 1.1% of the total initial spleen activity. Even allowing for some DC loss during enrichment, this is a very low recovery of activity. However, this final result is in line with the results in Tables 2 and 3, indicating that most CD8+ DC precursor activity did not segregate with individual markers for the CD8- fully developed DCs. Although this low-level production from the sorted CD8- DCs might point to one minor route of CD8+ DC production, it could also arise from a high-efficiency precursor contaminant. Rather than resolve this issue, we consider future investigation should focus on the major pathways leading to CD8+ (and CD8-) DCs. What, then, are the characteristics of most immediate precursors of the CD8+ DCs found in normal laboratory mice? We find they are lighter in density than most spleen lymphocytes but more dense than mature DCs, but at present we have little further information. One candidate precursor, the mouse plasmacytoid cell, has recently been eliminated because it produces a subtype of CD8+ DCs that differs in other markers and that only produces this CD8+ DC subtype after microbial stimulation.15 Our kinetic studies9,15 indicate that the 3 mature DC subsets and the plasmacytoid cells of normal mouse spleen represent separate development streams at least as far back as the last dividing precursor. However, this may not be very far back, and the separate streams could well branch off from a common DC-committed precursor.16 The relationship between all these DC subtypes and the nature and lineage commitment of their immediate precursors remain to be elucidated.    Footnotes   Submitted October 24, 2002; accepted March 10, 2003. Prepublished online as Blood First Edition Paper, March 20, 2003; DOI 10.1182/blood-2002-10-3186. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: Ken Shortman, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, 3050, Victoria, Australia; e-mail: shortman{at}wehi.edu.au.    References Top Abstract Introduction Materials and methods Results Discussion References   Shortman K, Liu Y-J. Mouse and human dendritic cell subtypes. Nat Rev Immunol. 2002;2: 153-163. Wu L, D'Amico A, Hochrein H, Shortman K, Lucas K. Development of thymic and splenic dendritic cell populations from different hemopoietic precursors. Blood. 2001;98: 3376-3382.[Abstract/Free Full Text] Manz MG, Traver D, Miyamato T, Weissman IL, Akashi K. Dendritic cell potentials of early lymphoid and myeloid progentitors. Blood. 2001;97: 3333-3341.[Abstract/Free Full Text] Aliberti J, Schulz O, Pennington DJ, et al. Essential role for ICSBP in the in vivo development of murine CD8+ dendritic cells. Blood. 2003;101: 305-310.[Abstract/Free Full Text] Guerriero A, Langmuir PB, Spain LM, Scott EW. PU.1 is required for myeloid-derived but not lymphoid-derived dendritic cells. Blood. 2000;95: 879-885.[Abstract/Free Full Text] O'Keeffe M, Hochrein H, Vremec D, Pooley J, Evans R, Woulfe S, Shortman K. Effects of administration of progenipoietin 1, Flt-3 ligand, granulocyte colony-stimulating factor, and pegylated granulocyte-macrophage colony-stimulating factor on dendritic cell subsets in mice. Blood. 2002;99: 2122-2130.[Abstract/Free Full Text] Wu L, D'Amico A, Winkel KD, Suter M, Lo D, Shortman K. RelB is essential for the development of myeloid-related CD8- dendritic cells but not of lymphoid-related CD8+ dendritic cells. Immunity. 1998;9: 839-847.[CrossRef][Medline] [Order article via Infotrieve] Wu L, Nichogiannopoulou A, Shortman K, Georgopoulos K. Cell-autonomous defects in dendritic cell populations of Ikaros mutant mice point to a developmental relationship with the lymphoid lineage. Immunity. 1997;7: 483-492.[CrossRef][Medline] [Order article via Infotrieve] Kamath A, Pooley J, O'Keeffe M, et al. The development, maturation and turnover rate of mouse spleen dendritic cell populations. J Immunol. 2000;165: 6762-6770.[Abstract/Free Full Text] Anjuere F, Martinez del Hoyo G, Martin P, Ardavin C. Langerhans cells acquire a CD8+ dendritic cell phenotype on maturation by CD40 ligation. J Leukoc Biol. 2000;67: 206-209.[Abstract] Merad M, Fong L, Bogenberger J, Engleman EG. Differentiation of myeloid dendritic cells into CD8-positive dendritic cells in vivo. Blood. 2000; 96: 1865-1872.[Abstract/Free Full Text] Martinez del Hoyo G, Martin P, Arias CF, Marin AR, Ardavin C. CD8+ dendritic cells originate from the CD8- dendritic cell subset by a maturation process involving CD8, DEC-205, and CD24 up-regulation. Blood. 2002;99: 999-1004.[Abstract/Free Full Text] Vremec D, Pooley J, Hochrein H, Wu L, Shortman K. CD4 and CD8 expression by dendritic cell subtypes in mouse thymus and spleen. J Immunol. 2000;164: 2978-2986.[Abstract/Free Full Text] Shortman K. Analytical and preparative equilibrium density separation of lymphoid cells on albumin and metrizamide. Methods Enzymol. 1984; 108: 102-117.[Medline] [Order article via Infotrieve] O'Keeffe M, Hochrein H, Vremec D, et al. Mouse plasmacytoid cells: long-lived cells, heterogeneous in surface phenotype and function, that differentiate into CD8+ dendritic cells only after microbial stimulus. J Exp Med. 2002;196: 1307-1319.[Abstract/Free Full Text] Martinez del Hoyo G, Martin P, Vargas HH, Ruiz S, Arias CF, Ardavin C. Characterization of a common precursor population for dendritic cells. Nature. 2002;415: 1043-1047.[CrossRef][Medline] [Order article via Infotrieve] CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: S. Sekine, K. Kataoka, Y. Fukuyama, Y. Adachi, J. Davydova, M. Yamamoto, R. Kobayashi, K. Fujihashi, H. Suzuki, D. T. Curiel, et al. A Novel Adenovirus Expressing Flt3 Ligand Enhances Mucosal Immunity by Inducing Mature Nasopharyngeal-Associated Lymphoreticular Tissue Dendritic Cell Migration J. Immunol., June 15, 2008; 180(12): 8126 - 8134. [Abstract] [Full Text] [PDF] Y. Xu, Y. Zhan, A. M. Lew, S. H. Naik, and M. H. Kershaw Differential Development of Murine Dendritic Cells by GM-CSF versus Flt3 Ligand Has Implications for Inflammation and Trafficking J. Immunol., December 1, 2007; 179(11): 7577 - 7584. [Abstract] [Full Text] [PDF] D. Vremec, M. O'Keeffe, H. Hochrein, M. Fuchsberger, I. Caminschi, M. Lahoud, and K. Shortman Production of interferons by dendritic cells, plasmacytoid cells, natural killer cells, and interferon-producing killer dendritic cells Blood, February 1, 2007; 109(3): 1165 - 1173. [Abstract] [Full Text] [PDF] J. Diao, E. Winter, C. Cantin, W. Chen, L. Xu, D. Kelvin, J. Phillips, and M. S. Cattral In Situ Replication of Immediate Dendritic Cell (DC) Precursors Contributes to Conventional DC Homeostasis in Lymphoid Tissue. J. Immunol., June 15, 2006; 176(12): 7196 - 7206. [Abstract] [Full Text] [PDF] J. K. H. Tan and H. C. O'Neill Maturation requirements for dendritic cells in T cell stimulation leading to tolerance versus immunity J. Leukoc. Biol., August 1, 2005; 78(2): 319 - 324. [Abstract] [Full Text] [PDF] S. H. Naik, A. I. Proietto, N. S. Wilson, A. Dakic, P. Schnorrer, M. Fuchsberger, M. H. Lahoud, M. O'Keeffe, Q.-x. Shao, W.-f. Chen, et al. Cutting Edge: Generation of Splenic CD8+ and CD8- Dendritic Cell Equivalents in Fms-Like Tyrosine Kinase 3 Ligand Bone Marrow Cultures J. Immunol., June 1, 2005; 174(11): 6592 - 6597. [Abstract] [Full Text] [PDF] K. M. Kerksiek, F. Niedergang, P. Chavrier, D. H. Busch, and T. Brocker Selective Rac1 inhibition in dendritic cells diminishes apoptotic cell uptake and cross-presentation in vivo Blood, January 15, 2005; 105(2): 742 - 749. [Abstract] [Full Text] [PDF] A. E. Morelli, A. T. Larregina, W. J. Shufesky, M. L. G. Sullivan, D. B. Stolz, G. D. Papworth, A. F. Zahorchak, A. J. Logar, Z. Wang, S. C. Watkins, et al. Endocytosis, intracellular sorting, and processing of exosomes by dendritic cells Blood, November 15, 2004; 104(10): 3257 - 3266. [Abstract] [Full Text] [PDF] This Article Abstract Full Text (PDF) All Versions of this Article: 2002-10-3186v1 102/2/601    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Naik, S. Articles by Shortman, K. Search for Related Content PubMed PubMed Citation Articles by Naik, S. Articles by Shortman, K. Related Collections Immunobiology Social Bookmarking