• Neonatal mice;
  • Flt3 ligand;
  • DC subpopulations;
  • Cytokine production


  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and method
  7. Acknowledgements

Treatment with the hematopoietic growth factor Flt3 ligand (FL) increases DC numbers in neonatal mice and enhances their resistance against intracellular pathogens. Flow cytometric analysis showed the presence of conventional DC (cDC) and plasmacytoid pre-DC (pDC) in neonatal spleens from untreated and FL-treated mice. CD8α and MHC class II expression on cDC and pDC was higher on DC from FL-treated mice than on DC from control littermates. After FL treatment, two additional subpopulations of DC-lineage cells were found that were able to produce IL-12 and IFN-α. The IL-12 production of cDC from FL-treated animals was more than 50-fold increased and their ability to stimulate T cell proliferation was also increased. We conclude that the enhanced resistance against intracellular pathogens was due to increased numbers of DC-lineage cells and their increased ability to produce the essential cytokines.


Ligand for fms-like tyrosine kinase 3, Flt3 ligand


Conventional DC


Plasmacytoid pre-DC

1 Introduction

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and method
  7. Acknowledgements

Neonates are very susceptible to intracellular pathogens and the high incidence of neonatal death is often caused by infections 1, 2. The immune system of neonates differs from that of adults in terms of quality and quantity of the different immune elements. Neonatal B cells lack appropriate costimulatory molecules, such as CD86 and CD80, and tend to transduce negative signals that inhibit immune responses or induce B cell apoptosis 2, 3. T cells in the fetus 4 as well as in neonates are functionally mature but present in reduced numbers 2, 59.

Dendritic cells (DC) also differ in neonates. DC are the key regulators of the immune response and provide the link between the innate and adaptive immune systems. They have the capacity of taking up pathogens 10, 11, of producing the appropriate cytokines for innate defence 1113, and of migrating to lymph nodes, where they present processed antigens to T cells, thus initiating adaptive immune response 11, 13. Data on the functional capacity of neonatal DC are controversial.In some reports neonatal DC are described as high producers of IL-12 14, 15, whereas the opposite is true in others 1618. The stimulus used to induce cytokine production appears to be a key variable 19. Overall it is not clear whether neonatal DC are able to produce sufficient amounts of IL-12 during infections in vivo. Neonatal DC are able to activate CD8 T cells 79, 20, but data on their efficiency compared to adult DC vary. Some reports indicate a deficiency in initiating T cell proliferative responses 21, while others suggest a capacity close to that of adult DC 17.

In adult mice, there are several distinct subpopulations of DC differing in surface phenotype and function. Conventional DC (cDC) display dendritic shape and express surface MHC class II and costimulatory molecules. They can be subdivided based on surface expression of other molecules, such as CD8α, CD4, CD11b and DEC-205 12, 2226. The CD4CD8α+CD11bDEC-205+ subtype includes the main producers of IL-12 and the CD4CD8αCD11b+DEC-205 subtype produces IFN-γ 12. Plasmacytoid pre-DC (pDC) are the main producers of IFN type I 2729. They are round cells, lacking DC morphology and having low surface expression of MHC class II and costimulatory molecules, and they are poor activators of T cells 3032. However, pDC resemble cDC after microbial stimulation 30, 31, 33, 34. The numbers of both cDC and pDC are elevated after treatment with the hematopoietic growth factor Flt3 ligand (FL) 23, 28, 35, 36.

FL causes proliferation of primitive precursor cells 37. FL treatment of adult mice causes an increase of DC, natural killer (NK) cells and B cells 38, 39, while treatment of neonatal mice leads to an increase of DC 40. In adult mice, FL treatment has been shown to lead to an increase in resistance against Herpes simplex virus type 1 (HSV-1) 41, Listeria monocytogenes42 and Leishmania43. Recently we have shown that FL treatment of newborn mice leads to an increased resistance against the intracellular pathogens HSV-1 and L. monocytogenes40. This was not only associated with an increase in the known DC subtypes, but there were also new DC subpopulations present, which had not been described before. Accordingly, we performed an extensive phenotyping of these FL-induced DC to study their state of maturation. Since IFN-α, -β and IL-12 44, 45 are essential for resistance against HSV-1 and L. monocytogenes, respectively 46, we also investigated the ability of these apparently different DC subpopulations to produce these cytokines.

2 Results

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and method
  7. Acknowledgements

2.1 FL treatment of neonatal mice increases the number of DC-lineage cells

Spleens of 7-day-old, untreated neonatal mice contained 200×106–400×106 cells, of which less than 1% (1.5×106) were CD11c+ DC. After treating newborn mice with FL for 7 days, the number of spleen cells stayed within this range, but the number of CD11c+ DC was increased to 4×106–5×106 cells per spleen. We first checked that these CD11c+ cells were indeed DC. After enrichment of DC-lineage cells, we confirmed that the selected cells were all negative for CD3, CD19 and DX5 (T cell, B cell and NK cell markers, respectively).

We then determined which DC subtypes were present within the FL-induced DC. To segregate cDC and pDC we stained for CD11c and CD45RA, since cDC have high levels of CD11c and no CD45RA or B220 expression, whereas pDC have an intermediate expression of CD11c, high expression of CD45RA, and are B220+33. In spleens of untreated 7-day-old mice, these two subpopulations were present (Fig. 1), with about 210,000 cDC and 120,000 pDC obtained per spleen after DC enrichment. These numbers were dramatically enhanced after FL treatment. The number of cDC increased tenfold to 2×106 cells/spleen, and the total number of pDC increased threefold to 400,000 cells/spleen (Table 1). Microscopy showed that all these FL-induced DC are rather round cells, lacking obvious DC morphology (data not shown). Thus, they appeared to be immature, comparable with DC precursors in adult mouse blood 47.

To determine the localization of the increased cDC and pDC, immunohistology of spleen, liver and bone marrow was performed. This complemented our previous study showing an increase in DC-lineage cells in the skin of FL-treated 7-day-old mice 40. After FL treatment, the number of CD11c-bearing cells in spleen and liver of neonatal mice increased (Fig. 2a), confirming the flow cytometry analysis. In bone marrow, however, the initially very low level of CD11c+ cells did not increase (data not shown). To distinguish between cDC and pDC, sections were also stained for B220. Although this would also stain B cells, we had determined by flow cytometry that there was no increase in CD19 staining B cells. In untreated neonates B220 staining was mainly found in the B cell areas in spleen and almost none was detected in liver (Fig. 2b). After FL treatment there was a remarkable increase in B220+ cells, mainly seen scattered around rather than in the B cell areas of spleen (Fig. 2b). Also there were many more cells, which were B220+, in liver (Fig. 2b). These cells are likely to be pDC, although earlier precursors of DC, NK cells and B cells could also contribute.

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Figure 1. Analysis of DC subpopulations in spleen of untreated and FL-treated neonatal and of adult mice. DC-enriched spleen cells from untreated and FL-treated neonatal mice and adult mice were analyzed by flow cytometry with antibodies against CD11c and CD45RA. One typical experiment out of six is shown.

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Table 1. Absolute and relative numbers of DC subpopulations and their fractions determined by CD8α and CD4 expression in spleen of untreated and FL-treated neonatal mice
 ×103a)%b)×103a)% b)Increase c)
  1. a) Absolute cell numbers/spleen.

  2. b) Relative cell numbers.

  3. c) Increase of absolute cell numbers.

cDCCD11chi/CD45RAneg210 2,000 10×
pDCCD11cint/CD45RAhi120 400 
New DC subpopulationCD11chi/ CD45RAintNot present 400 
 CD4CD8a+ 24060 
 CD4CD8a 16040 
New DC subpopulationCD11chi/CD45RAhiNot present 500 
  CD4CD8a+ 20040 
 CD4+CD8a 5010 
 CD4CD8a 25050 
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Figure 2. Immunohistology of spleen and liver from untreated and FL-treated neonatal mice. Cells were stained with antibodies against CD11c (a) and B220 (b) in spleen and liver of untreated and FL-treated mice.

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2.2 FL treatment of neonatal mice produces novel DC subpopulations

As well as the established cDC and pDC populations, the CD11c and CD45RA distribution plots (Fig. 1) indicated that there were additional DC populations present in 7-day-old mice after FL treatment. A small but distinct population showed high CD11c and intermediate CD45RA expression (CD11chiCD45RAint DC); there were 400,000 cells/spleen of this population. Another population expressed high levels of CD11c and CD45RA (CD11chiCD45RAhi DC); there were 500,000 cells/spleen of this population (Fig. 1, Table 1).

Previously described DC isolation protocols used mAb against B220 (CD45R) to deplete B cells 23. It was possible that the new B220+ DC subpopulations we have described in the neonate had not been observed in adult mice after FL treatment because they had been depleted along with B cells. Therefore, adult mice were also treated with FL to determine whether the new subpopulations were neonate specific. There were DC present with high expression of the surface markers CD11c and CD45RA (Fig. 1), although not as distinct as in neonates. Thus, this new subpopulation was not unique in FL-treated neonates. However, the CD11chiCD45RAint cells were not observed as a distinct subpopulation in adults after FL treatment (Fig. 1).

To check whether the CD11chiCD45RAhi DC were developmental stages of either pDC or cDC, they were sorted and cultured in FL overnight. Neither neonatal nor adult CD11chiCD45RAhi DC turned into pDC nor cDC (data not shown). Since they might need the appropriate environment in vivo for further development, 5- (and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled CD11chiCD45RAhi DC were transferred into FL-treated mice and re-analyzed after 36 h. They did not develop into cDC or pDC, but remained present as a distinct subpopulation of the same phenotype without any evident expansion in numbers (data not shown).

2.3 FL treatment induces changes in the surface phenotype of neonatal DC

The DC subpopulations in the FL-treated neonatal mice were analyzed by flow cytometry for expression of other surface markers, to determine whether they differed from the DC in untreated neonatal or adult mice. CD8α and CD4 were first tested, since they serve to delineate the DC subtypes in spleen. FL treatment led to a predominant increase in DC-lineage cells expressing CD8α (Fig. 3). Unlike the adult cDC, the major population (∼50%) of cDC from untreated neonates was CD8α+. After FL treatment the CD8α+ subset still predominated, constituting over 70% of the total cDC (Fig. 3a). Thus, the CD4CD8α+ cDC subset was increased 14-fold, whereas the CD4+CD8α and CD4CD8α DC subtypes increased only 5-fold. CD8α+ pDC (CD4 and CD4+) also became the predominant populations of pDC from FL-treated neonates. The CD4+CD8α+ pDC increased 5-fold, whereas the CD4+CD8α pDC increased 1.5-fold and the CD4CD8α pDC increased 3-fold (Table 1). Of the novel populations observed only after FL treatment, the CD11chiCD45RAint group contained 60% CD4CD8α+ cells, the remainder CD8αCD4 cells. The CD11chiCD45RAhi DC were 40% CD4CD8α+ and 10% CD4+CD8α. In both populations there were no CD4+CD8α+ cells detectable and thus they showed a CD8α and CD4 marker distribution similar to that of cDC (Fig. 3c, Table 1).

The interdigitating cell marker DEC-205 48 is usually strongly associated with CD8α-bearing cDC in adult mice and is not normally expressed on pDC 23. Because of the increased expression of CD8α on cDC from FL-treated neonatal mice, we examined whether CD8α expression was concomitant with DEC-205 expression (Fig. 4). In untreated neonatal mice, CD11cint cells, which included pDC (Fig. 4a, fraction A), were DEC-205. The cDC with a high expression of CD11c were divided into two distinct fractions, of DEC-205+ and DEC-205 cells (Fig. 4a, fractions B and C), the DEC-205+ cells being mostly CD8α+. Thus, the DC-lineage cells from untreated neonates co-expressed CD8α and DEC-205 in the same expression pattern as adult animals.

The expression of MHC class II, CD80 and CD86 was examined since these indicate the state of DC maturation or activation. The DC of neonatal mice have been reported to show only low expression of MHC class II 2, 5 but have expression levels comparable to adults by 2 weeks of age 17. We found that 55–65% of the cDC of untreated neonates were MHC class II+ (Fig. 3a) with a somewhat lower expression level than on adult cDC. After FL treatment, over 90% of the cDC were MHC class II+ and the level of expression was increased to adult levels (Fig. 3a and data not shown). MHC class II expression of pDC in neonates was very low, even lower than on pDC of adult mice (Fig. 3b and data not shown). After FL treatment, there were more MHC class II+ cells, but their expression level did not increase (Fig. 3b). The levels of activation markers such as CD80 and CD86 were not significantly influenced by FL treatment (data not shown). Thus, although the established DC subsets showed some increases in maturation after FL treatment, they could not be considered as activated DC.

Amongst the novel FL-induced DC subsets, the MHC class II expression was overall similar to that of the cDC in the same mice. However, it did serve to subdivide the novel subsets, although the significance of this is not clear. Thus, amongst the CD11chiCD45RAhi DC there were two fractions, one B220+MHC class II and the other B220+MHC class II+ (Fig. 3c, fraction A). CD11chiCD45RAint DC could be divided into three fractions using these markers. They contained both B220+ and B220 fractions (Fig. 3c, fraction B). The B220+ fraction expressed low to high levels of MHC class II, while the cells within the B220 fraction all expressed high levels of MHC class II (Fig. 3c, fraction B).

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Figure 3. Analysis of DC subpopulations in spleen of untreated and FL-treated neonatal mice. (a) cDC and (b) pDC from DC-enriched spleen cells of untreated and FL-treated neonatal mice and (c) CD11chiCD45RAhi DC (A) and CD11chiCD45RAint DC (B) from DC-enriched spleen cells from FL-treated neonatal mice were analyzed by flow cytometry with antibodies against CD8α, CD4, B220 and MHC class II. One typical experiment out of six is shown.

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Figure 4. Analysis of DC subpopulations in spleen of untreated and FL-treated neonatal mice. DC-enriched spleen cells from untreated controls (a) and FL-treated neonatal mice (b) were analyzed by flow cytometry with antibodies for CD11c and DEC-205. Gated cells (A–F) were further analyzed with antibodies against CD8α and CD4. One typical experiment out of six is shown.

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2.4 FL treatment results in increased cytokine production by DC of neonatal mice

As a test of the functional capacity of the DC populations induced by FL, their capacity to produce two key cytokines, IL-12 and IFN-α, was determined. Although data on IL-12 production by neonatal DC have been controversial 1419, our results were in accord with the view that there is no intrinsic defect 19. We found that after appropriate activation, bioactive IL-12p70 production by cDC from 7-day-old mice was moderate (90 pg/ml; Fig. 5a). However, the cDC induced by FL in neonatal mice showed a 50-fold increase in IL-12p70 to 5,800 pg/ml. This could not be explained by an increase in IL-12p70-producing CD8+ cDC, since these only increased from 50% to 75% of the cDC population (Fig. 3a).

The capacity of the novel FL-induced DC populations to produce IL-12p70 was determined, since they might be an additional source of this cytokine. CD11chiCD45RAint DC produced 6,000 pg/ml, about the same amount as produced by cDC of the FL-treated group (Fig. 5a). The CD11chiCD45RAhi DC also had the ability to produce IL-12p70 at a level of 3,600 pg/ml (Fig. 5a). While they were less efficient than cDC or CD11chiCD45RAint DC, they still produced 40 times more IL-12p70 than cDC of control neonates (Fig. 5a). We established that the CD11chiCD45Rahi DC of adult mice were also able to produce IL-12p70 (data not shown).

The production of IFN-α after viral stimulation was most potent by the pDC of 7-day-old mice (Fig. 5b), as it is in adult mice. The production of IFN-α was somewhat enhanced (almost 50%) with pDC from FL-treated neonates. Of the novel DC subpopulations, the FL-induced CD11chiCD45RAhi DC of neonatal as well as adult mice were able to produce IFN-α (Fig. 5b and data not shown).

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Figure 5. IL-12- and IFN-α-producing capacity of DC subpopulations from spleen of untreated and FL-treated neonatal mice. Sorted subpopulation of splenic DC (5×104/ml) from untreated controls (open bars) and FL-treated mice (solid bars) were stimulated in culture with CpG-ph, IL-4, IFN-γ and GM-CSF (a) or with HSV-1 (MOI 1) (b). Supernatants were harvested after overnight culture and analyzed by ELISA for production of IL-12p70 (a) and IFN-α (b). In the supernatant of unstimulated DC populations no cytokines were detectable. These experiments were performed at least three times.

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2.5 Neonatal cDC of FL-treated mice display an enhanced ability to stimulate T cells

To compare the T cell stimulation capacity of the DC from untreated neonatal mice with that of FL-treated mice, an allogenic mixed lymphocyte reaction (MLR) was used. The moderate ability of neonatal DC to stimulate thymidine uptake by allogenic T cells was enhanced in the DC from FL-treated mice (Fig. 6). The novel CD11chiCD45RAhi DC subset from FL-treated mice was also able to stimulate allogenic T cells, less efficiently than the cDC from the same mice at low DC input but with similar efficiency when higher DC numbers were used (Fig. 6).

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Figure 6. Stimulation capacity of DC subpopulations from untreated and FL-treated neonatal mice. Purified splenic DC subpopulations from untreated controls and FL-treated mice were tested for their ability to stimulate naive T cells in an allostimulatory MLR at day 5. CD11chiCD45RAhi DC, cDC from FL-treated animals (FL cDC) and cDC from untreated neonates (control cDC) were compared.

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3 Discussion

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and method
  7. Acknowledgements

Overall, the administration of FL to neonatal mice leads to an increase in the number of DC, an increase in DC subpopulations capable of producing IL-12 and IFN-α, and especially for cDC, an increase in the amount of cytokine each DC is able to produce. FL treatment also leads to an increase in the T cell-stimulatory capacity of cDC and the induction of novel CD11c+ cells that also share this DC-specific function. This should translate into a remarked increase in resistance to infections.

Particularly interesting are the novel DC subpopulations, because they share characteristics of both pDC and cDC. The CD11chiCD45RAint DC have a very high ability to produce IL-12 and in accordance with this most of these cells are CD8α+. The expression of B220 is only intermediate, if present at all, and 80% of the cells are MHC class II+, which fits best with a cDC subset. The fact that they are not observed as a distinct population in adult mice after FL treatment supports the idea that they belong to the population of CD8α+ cDC, which is more distinct in neonates than in adults after FL treatment.

The second novel subpopulation, the CD11chiCD45RAhi DC, appears to have unique features but also shares some properties with other DC populations. The B220 expression on CD11chiCD45RAhi cells is as high as on pDC, while the MHC class II expression is comparable to cDC from FL-treated mice. These cells have the ability to produce high levels of IL-12 and low levels of IFN-α, and they are potent T cell stimulators. Thus, the CD11chiCD45RAhi cells resemble cDC although they express high levels of CD45RA and B220 and also have some ability to produce IFN-α. Since these cells display a mixed cDC/pDC phenotype, we hypothesized that they were possibly a developmental stage of one of these cells. However, cultivation with FL or transfer into FL-treated mice did not alter their surface phenotype to more closely resemble either pDC or cDC. Thus, the CD11chiCD45RAhi cells most likely represent a new DC subpopulation induced with FL treatment, which has not been observed earlier in adult mice, because of depleting antibodies against B220 used during DC enrichment procedures.

As well as increasing overall DC numbers and producing novel DC subsets, FL treatment increased the functional activities of the DC on an individual cell basis. Antigen presentation capacity, as judged by MLR, is slightly increased in line with some increase in MHC class II expression.

Taken together all these factors indicate that the neonatal mice treated with FL have a distinct advantage over their untreated littermate controls 40. FL induced an increase in IL-12-producing cells and led to a 50-fold enhancement of IL-12 production by cDC. This increase in IL-12 production would be crucial in defence against Listeria infection in vivo and most likely explains the increased survival of FL-treated neonates infected with this pathogen 40. Similarly, an increase in IFN-producing pDC and other DC in the FL-treated neonate would be beneficial in defence against HSV-1. We hypothesize that this increase in IFN production, possibly in synergy with increased IL-12 production and T cell activation, leads to increased resistance of FL-treated neonates to infection with this virus. The relative roles of the rapid innate and slower adaptive systems will vary with the nature of the pathogen and the pathogen load, but it is clear that the effects of FL on the DC system could form the basis of the increase in resistance of neonates to infection.

4 Materials and method

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and method
  7. Acknowledgements

4.1 Mice

C57BL/6 mice were bred under specific pathogen-free conditions at the Labortierkunde, University of Zurich, or at the Walter and Eliza Hall Institute animal breeding facility. Litters were of mixed gender. The term neonatal mice refers to 7-day-old mice.

4.2 Treatment of mice with FL

Newborn mice were injected subcutaneously within 24 h after birth and for six consecutive days with 1 μg of human FL (Amgen, Seattle, WA) in 50 μl of phosphate-buffered saline (PBS). Adult mice were treated with 10 μg human FL for 7 days. The PBS was verified to be free of endotoxins.

4.3 Immunofluorescence labeling of DC

The mAb, the fluorescent conjugates and the multicolor labeling procedure have been described elsewhere 49. For sorting cDC and pDC from spleens, anti-CD11c (N418)-FITC conjugate and anti-CD45RA (clone 14.8)-PE conjugate (BD Bioscience) were used. For surface phenotype analyses, the following mAb were used: anti-CD11c (N418)-FITC or -Alexa, anti-CD45RA (clone 14.8)-PE,anti-CD8 (YTS169.4)-Cy5, anti-CD4 (GK1.5)-Alexa, anti-CD11b (M1/70)-PE, anti-DEC-205 (NLDC)-PE, anti-B220 (RA3–6B2)-Cy5, MHC class II (M5/114)-Cy5 or -Alexa, anti-CD86 (GL-1)-FITC, anti-CD80 (16–10A1)-FITC, anti-CD44 (IM7.81)-FITC and anti-CD43 (S7)-FITC.

4.4 Flow cytometric sorting and analysis

For sorting cDC and pDC populations, a MoFlo (Cytomation Inc.), DIVA or ARIA (Becton Dickinson) instrument was used. An auto-fluorescent pre-sort was always performed. This involved sorting the cells before staining with any antibodies, but after PI staining. Only those cells which were negative and which did not fluoresce in the FITC or PE channel were collected, yielding a preparation free of auto-fluorescent cells and dead cells. Most analyses were performed on a FACS Star PlusTM instrument (Becton Dickinson) as described previously 49, using four fluorescent channels for the immunofluorescent staining (FL1 for FITC, FL2 for PE, FL3 for Cy5, FL4 for Alexa), with the FL5 channel to exclude any residual PI-positive cells or auto-fluorescent cells.

4.5 DC isolation procedure

The procedure is described elsewhere 49. Spleens of 8 to 21 pups were digested with collagenase and DNAse, followed by EDTA treatment at room temperature. The next steps were performed at 4°C. Light-density cells were collected after density cut with Nycodenz 1.077 g/cm3 (Nycomed, Oslo, Norway). The cells were incubated with an antibody depletion cocktail, which contained anti-CD3 [KT3, anti-Thy1 (T24/31.7)], anti-CD19 (ID3), anti-GR-1 (RB6–8C5) and anti-erythrocyte (TER-119). Leaving out anti-GR-1 did not increase the yield or change the phenotype of the pDC recovered. 33. The DC-enriched cells were 50–90% CD11c+. For purification and segregation of the populations of cDC and pDC, the pre-sorted DC preparation, containing both cell types, was labeled with anti-CD11c (N418)-FITC and anti-CD45RA (clone 14.8)-PE, and the distinct populations were sorted using the MoFlo (Cytomation Inc.), DIVA or ARIA (Becton Dickinson) instrument. The sorted subpopulations were at least 95% pure.

4.6 Cytokines and stimulants of DC

Mouse recombinant (r) GM-CSF (used at 200 U/ml), mouse rIL-4 (100 U/ml) and human rFL (100 ng/ml) were gifts from Amgen. Rat rIFN-γ (used at 20 ng/ml and bioactive with mouse cells) was purchased from Pepro Tech (Australia). Oligonucleotides containing a fully phosphorylated CpG motif were synthesized by Gene Works Pty Ltd (Australia), according to a published sequence (CpG1668 50), and used at 1,000 nM. Stimulation with virus was performed with 1 multiplicity of infection (MOI) of HSV-1.

4.7 Quantitation of cytokine production

Analysis of IL-12p70 production in culture supernatants was performed by two-site ELISA as described previously 12, 51. IFN-α was assayed by a two-site ELISA, using anti-mouse IFN-α (clone RMMA-1; PBL Biomedical Laboratories) as a capture antibody, and as a detection antibody a rabbit anti-mouse IFN-α polyclonal antibody (PBL Biomedical Laboratories), followed by a F(ab′)2 fragment donkey anti-rabbit-horseradish peroxidase conjugate (Jackson ImmunoResearch Laboratories, West Grove, PA).

4.8 Immunohistology

Freshly removed organs were immersed in Hank's balanced salt solution and snap-frozen in liquid nitrogen. For the staining of cell differentiation markers, frozen tissue sections of 5 mm thickness were cut in a cryostat, placed on siliconized glass slides, air-dried, fixed in acetone for 10 min and stored at –70°C. Rehydrated tissue sections were incubated with primary rat mAb against CD45R/B220 (RA3–6B2; PharMingen) or anti mouse CD11c (PharMingen), and with primary hamster mAb (N418). Primary antibodies were revealed by sequential incubation with goat antibodies against species-specific Ig, followed by alkaline phosphatase-labeled donkey antibodies against goat Ig (Jackson ImmunoResearch). Dilutions of anti-Ig reagents were made in Tris-buffered saline containing 5% normal mouse serum. Alkaline phosphatase was visualized using naphthol AS-BI (6-bromo-2-hydroxy-3-naphtholic acid-2-methoxy anilide) phosphate and new fuchsine as substrate, yielding a red-colored reaction product. Endogenous alkaline phosphatase was blocked by levamisole. Color reactions were performed at room temperature for 15 min with reagents from Sigma Chemical Co. (St. Louis, MO). Sections werecounterstained with hemalum and coverslips mounted with glycerol and gelatin.

4.9 Mixed lymphocyte reaction

T cells were purified from pooled mesenteric, axillary, brachial and inguinal lymph nodes of adult CBA/J mice. Briefly, cell suspensions from lymph nodes were obtained by passing the organs through a fine mesh sieve. Cells were washed with PBS containing 2% FCS. The cells were then depleted of cells other than T cells by incubation with a mixture of the following antibodies: Ter119, RA3–6B2, M1–70 and RB6–6CS. Antibody-coated cells were removed by incubation with sheep anti-rat IgG magnetic beads (Dynal). The T cells were washed, suspended in culture medium (modified RPMI 1640 containing 10% FCS) and counted. A culture tray was incubated at 37°C in 10% CO2 in air for 6 days. On day 5 the culture tray was pulsed with [3H]thymidine [1 μCi (0.0185 MBq)/well] for 6 h and then frozen. The tray was thawed, the cells were washed and harvested on glass fiber filters and the amount of thymidine incorporated was measured using liquid scintillation. All cultures were done in triplicate and background controls with T cells only were included.

4.10 Transfer of CD11chiCD45RAhi DC

Sorted cells (2.5×106) were labeled with CFSE and transferred into FL-treated mice by i.v. injection. Thirty-six hours later, transferred DC were isolated and analyzed by flow cytometry for CD11c and CD45RA expression.


  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and method
  7. Acknowledgements

We thank V. Lapatis, D. Kaminaris, C. Tarlington and Frank Battye from The Walter and Eliza Hall Institute (WEHI) for their help with FACS analysis and sorting. Thanks also to David Vremec and Ben Fancke from the WEHI for maintaining an excellent infrastructure. Thanks also to Norbert Wey, Department of Pathology, University of Zurich, for his help with photography of the histology slides. This work was supported by a grant from the Swiss Science Foundation, by the Kanton of Zurich and by Bavarian Nordic, Munich, Germany.

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