FASEB J.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Published as doi: 10.1096/fj.05-5411fje.
This Article
Right arrow Abstract Freely available
Right arrow Summary
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
fj.05-5411fjev1
fj.05-5411fjev2
20/7/982    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chaudhry, U. I.
Right arrow Articles by DeMatteo, R. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chaudhry, U. I.
Right arrow Articles by DeMatteo, R. P.
(The FASEB Journal. 2006;20:982-984.)
© 2006 FASEB

In vivo overexpression of Flt3 ligand expands and activates murine spleen natural killer dendritic cells

Umer I. Chaudhry, Steven C. Katz, T. Peter Kingham, Venu G. Pillarisetty, Jesse R. Raab, Alaap B. Shah and Ronald P. DeMatteo1

Hepatobiliary Service, Memorial Sloan-Kettering Cancer Center, New York, New York, USA

1Correspondence: Memorial Sloan-Kettering Cancer Center, Box 203, 1275 York Ave., New York, NY 10021 USA. E-mail: dematter{at}mskcc.org

ABSTRACT

Natural killer dendritic cells (NKDC) are a unique class of murine immune cells that possess the characteristics of both natural killer (NK) cells and dendritic cells (DC). Because NKDC are able to secrete IFN-{gamma}, directly lyse tumor cells, and present antigen to naïve T cells, they have immunotherapeutic potential. The relative paucity of NKDC, however, impedes their detailed study. We have found that in vivo, overexpression of the hematopoietic cytokine Flt3 ligand (Flt3L) expands NKDC in various organs from 2–18 fold. Flt3L expanded splenic NKDC retain the ability to lyse tumor cells and become considerably more potent at activating naïve allogeneic and antigen-specific T cells. Compared to normal splenic NKDC, Flt3L-expanded splenic NKDC have a more mature phenotype, a slightly increased ability to capture and process antigen, and a similar cytokine profile. In vivo, we found that Flt3L-expanded splenic NKDC are more effective than normal splenic NKDC in stimulating antigen-specific CD8 T cells. Additionally, we show that NKDC are able to cross-present antigen in vivo. The ability to expand NKDC in vivo using Flt3L will facilitate further analysis of their unique biology. Moreover, Flt3L-expanded NKDC may have enhanced immunotherapeutic potential, given their increased ability to stimulate T cells.—Chaudhry, U. I., Katz, S. C., Kingham, T. P., Pillarisetty, V. G., Raab, J. R., Shah, A. B., and DeMatteo, R. P. In vivo overexpression of Flt3 ligand expands and activates murine spleen natural killer dendritic cells.


Key Words: antigen presentation/processing • cell proliferation • cytotoxicity

NATURAL KILLER DENDRITIC CELLS (NKDC) are a unique class of murine immune cells that express both the natural killer (NK) cell marker NK1.1 and the dendritic cell (DC) marker CD11c. We recently reported that NKDC are present in the spleen, lymph node, thymus, and liver of normal mice (1) . NKDC can directly lyse tumor cells, capture tumor-derived antigen, and subsequently activate naive antigen-specific T cells in vitro. Thus, they may link innate and adaptive immunity in vivo in mice. On activation with CpG and interleukin (IL)-4, NKDC secrete high levels of IFN-{gamma}, while NK cells and DC produce negligible amounts. We have shown that NKDC secretion of IFN-{gamma} depends on autocrine IL-12 (1) . Because of their pleiotropic functions, NKDC appear to have immunological importance.

Using anti-CD11c immunomagnetic bead enrichment and fluorescence-activated cell sorting (FACS), we have been able to isolate ~4–7 x 104 NKDC per mouse spleen. Although this yield has allowed for preliminary in vitro experiments, the relative scarcity of NKDC remains an impediment to the further study of their unique immunobiology and the assessment of their therapeutic potential in vivo. The situation is even more problematic than the difficulty of studying freshly isolated murine DC before the use of cytokines to expand them in vivo (2 3 4 5 6) , as NKDC constitute only a small percentage (4–5%) of total splenic DC. Thus, a means to expand NKDC either in vivo or in vitro is desirable.

Flt3 ligand (Flt3L) is a hematopoietic cytokine that has been shown to expand myeloid and lymphoid progenitor cells, as well as DC and NK cells (2 ,3 ,7 ,8) . Flt3L has also been shown to enhance DC immune function in a variety of mouse models. Systemic administration of Flt3L can restrict tumor growth, prolong survival of animals harboring tumors, and even mediate regression or stabilization of established tumors (9 10 11 12) . We and others have found that in vivo, overexpression of Flt3L generates DC that may have tolerogenic effects on T cells (3 ,13 ,14) . Because Flt3L expands DC in vivo and, to a lesser extent, NK cells, we hypothesized that it would also expand NKDC.

We have found that in vivo overexpression of Flt3L expands the number of NKDC from 2–18-fold in both lymphoid and nonlymphoid organs. Furthermore, Flt3L-expanded splenic NKDC have potent cytolytic activity and increased T cell stimulatory capacity in vitro. In vivo, Flt3L-expanded splenic NKDC stimulate antigen-specific T cells to a considerably greater extent than do normal NKDC. Because of their increased capacity to elicit a T cell immune response, Flt3L-expanded NKDC are an attractive candidate for cellular immunotherapy.

MATERIALS AND METHODS

Mice
Adult 6- to 8-wk-old male C57BL/6 (B6, H-2Kb) mice were purchased from Taconic Farms (Germantown, NY). Rag2-/-OT-I (OT-I), Rag2-/-OT-II (OT-II) OVA TCR transgenic, and CD45.1 mice, each on a B6 background, were also obtained from Taconic. Balb/c (H-2Kd) mice were acquired from Jackson Laboratories (Bar Harbor, ME). Mice were maintained in the pathogen-free animal housing facility at Memorial Sloan-Kettering Cancer Center, and all procedures were approved by the Institutional Animal Care and Use Committee.

Cell isolation
Mice were administered a single tail vein injection of 1 x 109 plaque forming units (pfu) of a recombinant adenovirus vector that we constructed previously (3) , which carries the murine Flt3L gene (Flt3L). Control mice received either normal saline (NS) or a similar dose of an adenovirus encoding the alkaline phosphatase transgene (Ad). Liver and lung nonparenchymal cells were isolated as described previously (1 ,3 ,15) . Briefly, animals were euthanized by CO2 inhalation prior to a laparotomy and thoracotomy. The portal vein and right ventricle were cannulated in situ with a 26 1/2-gauge needles (BD Biosciences, San Diego, CA) and perfused with 2 ml of 1% collagenase IV (Sigma-Aldrich, St. Louis, MO) in PBS. Livers and lungs were mechanically disrupted and incubated in collagenase for 20 min at 37°C before being passed through a sterile 100-µm nylon mesh filter (BD Falcon, Franklin Lakes, NJ). Hepatocytes and pneumocytes were excluded by discarding the pellets from a series of three low-speed centrifugations (30 g x 5 min). Red blood cells were lysed using a hypotonic solution, and the resulting cell suspension was washed in media (RPMI 1640, 10% FCS, 2 mM DL-glutamine, 0.1% 2-mercaptoethanol, 100 µg/ml penicillin, 100 µg/ml streptomycin). Spleens, lymph nodes, and thymus were mechanically disrupted before being passed through a sterile 70-µm nylon mesh filter (BD Falcon). The resulting cell suspension was then pelleted (300 g x 7 min), red cells were lysed, and the remaining cells were washed twice in media. Splenocytes were separated into CD11c+ and CD11c– fractions with immunomagnetic beads per the manufacturer’s protocol (Miltenyi Biotech, Auburn, CA). Fc{gamma} receptors were blocked with the monoclonal antibody (Ab) 2.4G2 (Fc{gamma}III/IIR block; 1 µg/million cells; Monoclonal Antibody Core Facility, Sloan-Kettering Institute, New York, NY) before all immunomagnetic bead incubations. Enriched CD11c+ cells were stained with fluorescently conjugated antibodies to B220, NK1.1, and CD11c (all BD Pharmingen) for further separation of DC subtypes using a MoFlo cell sorter (DakoCytomation, Fort Collins, CO). Dead cells were excluded with 4',6-diamidino-2-phenylindole, dilactate (4',6'-diam idino-2-phenylidole, Molecular Probes, Eugene, OR). NKDC were defined as NK1.1+CD11c+ and DC as NK1.1–CD11c+B220– (thereby excluding B220+ plasmacytoid DC). NK cells were purified as NK1.1+CD11c–CD3– cells from the CD11c– fraction. Care was taken to exclude highly autofluorescent cells during FACS, and sorted cell populations were consistently >97% pure for the desired set of surface markers.

Flow cytometry
Four-color flow cytometry was performed on a FACSCalibur flow cytometer (BD Biosciences). Voltages were determined using unstained cells. Single-stained positive controls for each fluorochrome were used to set compensation. Samples were incubated with Fc{gamma}III/IIR block before staining. Approximately 5 x 105 cells were labeled with 0.1 µg fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll-a protein (PerCP), allophycocyanin (APC), or biotin conjugated Ab (all BD Pharmingen). Biotinylated antibodies were secondarily stained with streptavidin-PerCP. Cells were stained for CD45R/B220 [RA3–6B2], NK1.1 [PK136], CD11c [HL-3], MHC class I (H-2Kb) [AF6–88.5], MHC class II (I-Ab) [AF6–120.1], CD40 [3/23], CD80 [16–10A1], CD86 [GL-1], CD8 [53–6.7], CD3{epsilon} [145–2C11], and CD45.2 [104]. Appropriate immunoglobulin isotype controls were used for phenotype analysis. Flow cytometry data were analyzed with FlowJo software (TreeStar, Ashland, OR).

Cytokine secretion
Cytokine production was assessed by culturing 3 x 104 purified NKDC in a 96-well U-bottom tissue culture plate (BD Falcon) in 100 µl of media for 72 h. Supernatant IFN-{gamma}, TNF-{alpha}, IL-6, IL-10, and IL-12 concentrations were assayed using a cytometric bead array per the manufacturer’s protocol (BD Biosciences). The TLR 3 agonist poly (I:C) (10 µg/ml; Roche Applied Sciences, Chicago, IL), TLR 4 agonist LPS (10 µg/ml; Chemicon International, Inc., Temecula, CA), TLR 5 agonist flagellin (10 µg/ml; Calbiochem, San Diego, CA), TLR 7 agonist loxoribine (Loxo, 10 µg/ml; InvivoGen, San Diego, CA), or TLR 9 agonist CpG ODN 1826 (CpG, 10 µg/ml; Oligos Etc., Inc., Wilsonville, OR) were added to some wells.

Antigen uptake
To measure antigen uptake and processing, bulk DC were incubated in 150 µl of media with DQ-OVA (50 µg/ml, Molecular Probes, Eugene, OR) in duplicates at 37°C. At 0, 5, 15, and 30 min, the reactions were stopped by washing 3 times with ice-cold PBS before measurement of mean fluorescence on a FACSCalibur flow cytometer (BD Biosciences). NKDC were analyzed by gating on NK1.1+CD11c+ cells.

In vitro lytic and T cell assays
Lysis assays were performed using FACS purified splenic NK cells, NKDC, and DC. Cells were cocultured with 1 x 103 [51Cr] sodium chromate (PerkinElmer, Life and Analytical Sciences, Inc., Boston, MA) labeled Yac-1 cells (American Type Culture Collection, Manassas, VA) for 6 h in 96-well V-bottom plates (BD Falcon) in a total of 200 µl of media. [51Cr] sodium chromate release was measured with a TopCount NXT microplate scintillation and luminescence counter (PerkinElmer). Spontaneous release (no effectors) and maximum release (2% Triton-X; Sigma) were also assayed. Percent specific lysis was calculated as (cpm experimental – cpm spontaneous release) x 100/(cpm maximum release – cpm spontaneous release). Mixed leukocyte reactions (MLRs) were performed by combining stimulator cells with allogeneic T cells. T cells were purified using Thy1.2 [CD90.2] immunomagnetic beads (Miltenyi) according to the manufacturer’s protocol. FACS purified NK cells, NKDC, and DC from B6 mice were added in various numbers to 1 x 105 Balb/c T lymphocytes in 96-well U-bottom plates (BD Falcon) in a total of 200 µl of media. Antigen-specific T cell activation was assayed in a similar fashion with OT-I CD8+ transgenic T cells specific for SIINFEKL peptide (OVA257–264) (16) , or OT-II CD4+ transgenic T cells specific for KISQAVHAAHAEINEAG peptide (OVA323–339) (17) . Stimulators were loaded with the appropriate OVA peptide (1 µg/ml; Peptide Synthesis Core, Sloan-Kettering Institute) and plated at various concentrations with OT-I or OT-II T cells (3 x 104 per well) in a 96-well U-bottom plate. On day 3, the cultures were pulsed with 3H-thymidine (1 µCi/well, PerkinElmer), and radioactive uptake was measured 20 h later with a TopCount NXT microplate scintillation and luminescence counter (PerkinElmer).

In vivo T cell assay
T cells were isolated from the spleens of OT-I mice (CD45.2) using Thy1.2 immunomagnetic beads (Miltenyi) and subsequently incubated with 50-µM carboxy-fluorescein diacetate, succinimidyl ester (CFSE, Molecular Probes) at room temperature for 3 min. The cells were then washed 3 times with 5% FBS and once with PBS and resuspended in sterile normal saline. 3 x 106 CFSE-labeled OT-I T cells were injected into the lateral tail veins of CD45.1 mice. The following day, FACS-purified DC, NS NKDC, and Flt3L-expanded NKDC were loaded with SIINFEKL peptide (1 µg/ml) or ovalbumin protein (2 mg/ml) for 1 h at 37°C and washed 3 times with PBS. 5 x 104 loaded NKDC, in 50 µl of normal saline were injected into the footpads of CD45.1 mice that had received CFSE-labeled OT-I T cells 1 day earlier. Three days later, popliteal nodes were harvested, and single-cell suspensions were prepared. Nodal cells were analyzed for CD3, CD8, and CD45.2 expression. Proliferation of adoptively transferred OT-I T cells was measured by the dissipation of CFSE fluorescence. Nonviable cells were excluded from the analysis by using 7-aminoactinomycin D (7-AAD).

RESULTS

NKDC: a distinct subset of CD11c+ cells in murine lymphoid and nonlymphoid organs
We have recently identified a unique subset of cells expressing both the NK cell marker NK1.1, as well as the DC marker CD11c, in the spleen, lymph node, thymus, and liver of normal mice (1) . Because NKDC represent only 0.7% of splenocytes, it is advantageous to use cell enrichment before phenotype analysis or FACS purification. After enriching splenocytes with anti-CD11c immunomagnetic beads, NKDC are identified by gating on CD11c+ cells (Fig. 1 A) and using NK1.1 to distinguish them from conventional DC (Fig. 1B ). In the spleen, NKDC account for ~4–5% of total CD11c+-enriched cells. When compared to conventional DC, NKDC exhibit lower expression of CD11c (Fig. 1C ).


Figure 1
View larger version (23K):
[in this window]
[in a new window]
  		Figure 1. A distinct population of NKDC is present in the spleen.

A) Spleens were enriched for CD11c positive cells using immunomagnetic beads and positive selection columns. CD11c+ cells were gated by flow cytometry and analyzed for NK1.1 expression. B) NKDC were distinct from conventional DC (NK1.1–CD11c+B220–). C) When comparing CD11c expression, NKDC express lower levels of CD11c than conventional DC. Data are representative of 10 separate experiments with similar results.

In vivo overexpression of Flt3L expands NKDC
Because of the relative paucity of NKDC and difficulty of their isolation, our initial studies consistently required 20–30 mice per experiment to procure enough cells for in vitro analysis. Because Flt3L is a hematopoietic cytokine that expands DC and, to a lesser extent, NK cells, we hypothesized that Flt3L would also expand NKDC. To test this, we used an adenoviral vector encoding the cDNA for murine Flt3L to overexpress this cytokine in mice. We have previously used this vector to expand bulk DC in the mouse spleen (3) and shown that a single dose of the vector causes the serum concentration of Flt3L to peak at 1000 ng/ml on day 3 and then quickly drop by day 14 but remain elevated through day 28 (3) .

To determine the time course for optimal NKDC expansion, we injected mice with a single dose of 1 x 109 pfu of the Flt3L adenovirus vector and analyzed the number of splenic NKDC at serial time points. We found that in vivo overexpression of Flt3L leads to marked NKDC expansion (Table 1 ). Consistent with our previous findings on the effect of Flt3L overexpression on bulk splenic DC, NKDC expansion also peaked at day 10 (Fig. 2 A). The magnitude of the increase was almost fivefold over baseline, from ~6.0 x 105 cells to 3.0 x 106 cells, although NKDC represented only 0.5% of total splenocytes (Fig. 2A , Table 1 ). In contrast, splenic NK cells and DC underwent a 1.6- and 16-fold increase, respectively. Mice injected with an Ad control vector, which expressed an irrelevant transgene, experienced a 1.3-fold increase in NK cells, a 1.9-fold increase in NKDC, and a 1.8-fold increase in DC on day 10 (Fig. 2B-D ). In other lymphoid and nonlymphoid organs of mice treated with Flt3L, the increase in NKDC ranged anywhere from twofold in the thymus to 18-fold in the lung (Table 1) . After day 10, the effects of Flt3L on splenic NKDC expansion started to wane, and their number returned to baseline by day 35 (Fig. 2A ). As we obtained the greatest number of NKDC from the spleens of mice treated for 10 days with Flt3L (Table 1) , we performed the remaining experiments with these cells. After immunomagnetic bead enrichment and further purification by FACS, ~7 x 104 NKDC per normal spleen and 5 x 105 NKDC per Flt3L treated spleen were routinely isolated.


View this table:
[in this window]
[in a new window]
  		Table 1. Frequency of Flt3L-expanded natural killer dendritic cells


Figure 2
View larger version (14K):
[in this window]
[in a new window]
  		Figure 2. In vivo overexpression of Flt3L expands NKDC.

The time course of splenic NKDC expansion by Flt3L was determined by injecting 1 x 109 pfu of viral particles intravenously and assessing the numbers of NK1.1+CD11c+ splenocytes at serial intervals. A) NKDC population peaked at day 10 to 3 x 106 cells, and returned to baseline by day 35 (x-axis not to scale). B–D) When comparing day 10 expansion of splenic NK cells, NKDC, and DC, NKDC expansion was intermediate (fivefold) to that of NK cells (1.6-fold) and DC (16-fold). In contrast, mice injected with an Ad control vector experienced a 1.3-fold increase in NK cells, a 1.9-fold increase in NKDC, and a 1.8-fold increase in DC population on day 10. Data represent means of 2 mice per time point.

Flt3 ligand-expanded NKDC have similar cytolytic function
Because Flt3L is known to enhance the immune function of expanded DC (2 ,3 ,7) , we tested the functional characteristics of Flt3L-expanded splenic NKDC. To assess their cytolytic capacity, we used a standard chromium release assay. We found that Flt3L-expanded splenic NKDC maintained their ability to lyse Yac-1 targets (Fig. 3 A). They accomplished a similar degree of lysis as normal splenic NKDC and NK cells. As expected, conventional splenic DC did not lyse Yac-1 targets (not shown).


Figure 3
View larger version (15K):
[in this window]
[in a new window]
  		Figure 3. Flt3L-expanded splenic NKDC lyse tumor cells and induce greater proliferation of naïve and antigen-specific T cells.

A) FACS-purified NK cells, NKDC, and DC were added in various numbers to 1 x 103 Yac-1 cells that had been previously labeled with 51Cr (100 µCi/2 x 106 cells for 90 min). Six hours later, supernatants were harvested and analyzed in a gamma counter. B) Saline (NS), Ad, and Flt3L-expanded splenic NKDC were FACS-purified and an MLR was performed by incubating various numbers of stimulator cells with 1 x 105 allogeneic (Balb/c) splenic T cells that had been isolated with anti-Thy1.2 immunomagnetic beads. Alternatively, the antigen-specific T cell stimulatory capacity of NS control or Flt3L-expanded splenic NKDC was assessed by culturing them with OT-I CD8+ T cells (3 x104) and SIINFEKL peptide (1 µg/ml) (C) and OT-II CD4+ T cells (2 x 104) and KISQAVHAAHAEINEAG peptide (1 µg/ml) (D). In both allogeneic and antigen-specific assays, proliferation was measured by [3H]-thymidine uptake. Data are representative of three separate experiments with similar results.

Flt3 ligand-expanded NKDC have increased antigen-presenting function in vitro
After establishing that Flt3L-expanded splenic NKDC retained the ability lyse Yac-1 targets, we wanted to know whether they also had preserved T cell stimulatory capability. We first measured the ability of Flt3L-expanded splenic NKDC to activate allogeneic T cells in an MLR. We found that freshly sorted, Flt3L-expanded splenic NKDC had greater capability to activate naïve allogeneic T cells to proliferate than normal splenic NKDC (Fig. 3B ). The higher MLR seen with Flt3L-expanded splenic NKDC was not due to nonspecific effects from the adenovirus vector. Because the control Ad vector only slightly expanded NKDC (Fig. 2C ) and did not alter their T cell stimulatory function (Fig. 3B ), we chose to perform the rest of our T cell experiments just using splenic NKDC from mice injected with normal saline as a control.

To determine the effect of Flt3L expansion on the ability of NKDC to mediate antigen-specific T cell responses, we assessed their capacity to activate T cell receptor (TCR) transgenic CD8 and CD4 T cells. Flt3L-expanded splenic NKDC were cocultured with T cells isolated from OT-I and OT-II transgenic mice. Similar to the MLRs, we observed that Flt3L-expanded splenic NKDC were more potent stimulators of antigen-specific CD8 and CD4 T cell proliferation (Fig. 3C-D ).

Flt3 ligand-expanded NKDC are more mature
To determine why Flt3L-expanded NKDC induced greater T cell stimulation, we assayed the classic markers of DC maturation. Normal splenic NKDC displayed high expression of MHC I, low expression of MHC II, CD80, and CD86, and almost no expression of CD40. In contrast, we found that Flt3L-expanded splenic NKDC had increased expression of all the maturation markers tested (Fig. 4 ). The considerable increase in maturation was not due to the adenoviral vector alone, as Ad control splenic NKDC had only slight increases in MHC II, CD40, CD80, and CD86 expression, while there was no change in MHC I expression. As expected, DC had high expression of MHC II and costimulatory molecules.


Figure 4
View larger version (39K):
[in this window]
[in a new window]
  		Figure 4. Freshly isolated Flt3L-expanded NKDC are more mature than normal NKDC.

Cell surface expression of classic DC maturation markers was analyzed by flow cytometry. Shaded areas represent surface expression of the indicated markers. Isotype controls are shown as open histograms. Splenic DC are shown for comparison. Data are representative of five separate experiments with similar results.

Flt3 ligand-expanded NKDC have increased antigen uptake and processing
Another potential mechanism by which NKDC may become more immunogenic after Flt3L expansion is through enhanced antigen uptake and processing. To determine the antigen uptake and processing capacity of Flt3L-expanded splenic NKDC, we incubated bulk splenic DC with DQ-ovalbumin and measured mean fluorescence of NK1.1+CD11c+ cells at 0, 5, 15, and 30 min. DQ-ovalbumin requires proteolytic cleavage in order to become fluorescent, and thus it measures antigen uptake and a component of antigen processing. When compared to NS NKDC, we found that Flt3L-expanded splenic NKDC were slightly more efficient at antigen uptake and processing (Fig. 5 ).


Figure 5
View larger version (16K):
[in this window]
[in a new window]
  		Figure 5. Flt3L-expanded NKDC are slightly more efficient at antigen uptake and processing.

In vitro antigen uptake and processing assay was performed by incubating 1 x 106 freshly isolated bulk splenic CD11c+ cells with DQ-OVA for various times. Reactions were quenched with cold PBS and analyzed for mean fluorescence by flow cytometry. NKDC were analyzed by gating on NK1.1+CD11c+ cells. Data are representative of three separate experiments.

Flt3 ligand-expanded NKDC have a similar cytokine profile
To further investigate the potential factors responsible for the increased antigen-presenting function of Flt3L-expanded NKDC, we tested whether their baseline cytokine profile was altered. Similar to NS splenic NKDC, Flt3L-expanded splenic NKDC did not spontaneously secrete IFN-{gamma}, TNF-{alpha}, IL-6, or IL-10 in culture. As it is known that DC and NK cells are responsive to a variety of TLR ligands (18 19 20) , we tested the response of NKDC to poly (I:C) (TLR 3), LPS (TLR 4), flagellin (TLR 5), Loxo (TLR 7), and CpG (TLR 9). When stimulated with TLR ligands, Flt3L-expanded splenic NKDC secreted similar amounts of IFN-{gamma}, TNF-{alpha}, and IL-10 as NS splenic NKDC, except they secreted more IL-6 (Fig. 6 ). The IL-12 production for both cell types was low and close to the sensitivity concentration of the assay (10.7 pg/ml). Both normal and Flt3L-expanded splenic NKDC did not respond to stimulation by poly (I:C) or flagellin (not shown). Because it is known that DC can secrete IL-2 (21 ,22) , which is a potent T cell activator, we also tested the ability of unstimulated and stimulated splenic NKDC to secrete IL-2. We found that normal and Flt3L-expanded NKDC did not produce detectable levels of IL-2 in an unstimulated state or when activated with TLR agonists (not shown).


Figure 6
View larger version (12K):
[in this window]
[in a new window]
  		Figure 6. Saline and Flt3L-expanded splenic NKDC have a similar cytokine profile.

FACS-purified NS control and Flt3L- expanded splenic NKDC were cultured in 96-well plates at 3 x 104 cells per well in 100 µl for 72 h. Supernatant IFN-{gamma}, TNF-{alpha}, IL-6, IL-10 and IL-12 concentration were assayed by cytometric bead array. Various TLR agonists were added to some wells. Data are representative of two separate experiments with similar results.

Flt3 ligand-expanded NKDC have increased T cell stimulatory capacity in vivo
Because Flt3L-expanded NKDC had enhanced antigen-presenting function in vitro (Fig. 3B-D ), we wanted to determine whether their ability to stimulate T cells would be augmented in vivo. To test this, we used a well-established mouse footpad model. We adoptively transferred CFSE-labeled OT-I T cells (CD45.2+) into CD45.1+ mice. The following day we injected the footpads of the mice with NKDC that had been loaded with SIINFEKL peptide. Three days later, the draining popliteal lymph nodes were analyzed by flow cytometry. When gating on viable nodal cells, we found that mice treated with Flt3L-expanded splenic NKDC induced greater proliferation of adoptively transferred OT-I T cells (CD8+CD45.2+CFSE+), as measured by CFSE dissipation (Fig. 7 A), than mice treated with normal NKDC.


Figure 7
View larger version (23K):
[in this window]
[in a new window]
  		Figure 7. Flt3L-expanded splenic NKDC are more potent stimulators of antigen-specific T cells in vivo.

3 x 106 CFSE-labeled Thy1.2+ spleen OT-I T cells were injected in the tail veins of CD45.1 mice. The following day, FACS-purified NKDC were loaded with (A) SIINFEKL peptide or ovalbumin protein and injected into the footpads of CD45.1 mice. Three days later, popliteal nodes were harvested and the nodal cells were analyzed by flow cytometry. Proliferation of viable adoptively transferred OT-I T cells (CD8+CD45.2+) was determined by measuring CFSE fluorescence. Control mice that received OT-I T cells but no antigen-presenting cells are shown as open histograms. B) Flt3L-expanded NKDC were approximately one-half as potent as conventional DC at cross-presenting antigen while normal NKDC were only one-third as effective. Dead cells were excluded from the analysis using 7-AAD staining. Data are representative of two repetitions with similar results.

It is well accepted that DC are capable of cross-presenting antigen to CD8 T cells. To determine whether NKDC are able to cross-present antigen in vivo, we repeated the footpad experiment using freshly isolated DC, normal NKDC and Flt3L-expanded NKDC loaded with ovalbumin protein instead of SIINFEKL peptide. The cells were not treated with an exogenous stimulus before footpad inoculation. We observed that NKDC were able to take up ovalbumin protein and cross-present it to adoptively transferred OT-I T cells (Fig. 7A ). Once again, Flt3L-expanded splenic NKDC induced greater proliferation and accumulation of transgenic T cells when compared to normal NKDC. No proliferation was seen when cells were loaded with an irrelevant protein instead of ovalbumin (data not shown). When compared to conventional splenic DC, normal NKDC were approximately one-third as efficient at cross-presentation, as measured by the total number of CFSE+ T cells isolated from the popliteal nodes, while Flt3L-expanded NKDC were approximately one-half as potent as conventional splenic DC (Fig. 7B ).

DISCUSSION

NKDC are a unique class of murine immunological cells that share phenotypic and functional properties with both NK cells and DC (1) . NKDC are present in both lymphoid and nonlymphoid organs of normal mice. Although NKDC are rare cells (0.7% of total splenocytes), their ubiquitous distribution and pleiotropic functions suggest that they are physiologically important. Previously, we showed that NKDC can lyse tumor cells and present antigen to T cells. Although several groups have reported that DC produce IFN-{gamma} (23 24 25 26 27 28 29 30) , we have found that activated NKDC secrete substantial amounts of IFN-{gamma} while NK cells and DC make minimal amounts (1) . Furthermore, a recent paper by Schleicher et al. (31) demonstrates that IFN-{gamma} production previously ascribed to macrophages actually originates from either CD11b+CD11c+CD31+DX5+NK1.1+ NK cells or CD3+CD8+TCRß+ T cells contamination. Thus, NKDC are attractive for potential therapeutic manipulation, yet their analysis is constrained by their scarcity.

We now show that Flt3L expands NKDC in the spleen, liver, lungs, thymus, and lymph nodes of normal mice (Table 1) . The kinetics of expansion were similar to what we (3) and others (2 ,7) have previously observed with in vivo DC expansion after Flt3L treatment. Specifically, splenic NKDC expansion peaked at day 10 and remained elevated for up to 35 days (Fig. 2A ). The magnitude of expansion ranged anywhere from twofold in the thymus to 18-fold in the lung. We found the greatest absolute number of NKDC in the spleen, where they increased from a baseline of ~6.0 x 105 cells to an average of 3.0 x 106 cells, and as high as 5.0 x 106 cells depending on the experiment. When comparing expansion of splenic NK cells, NKDC, and DC, we observed that NKDC expansion was intermediate (fivefold) to that of NK cells (1.6-fold) and DC (16-fold) (Fig. 2B-D ).

While Flt3L-expanded splenic NKDC retained the potent cytolytic property of N splenic NKDC (Fig. 3A ), they induced considerably greater stimulation of naïve and antigen-specific T cell, both in vitro and in vivo (Figs. 3B-D , 7A and B ). The increased capacity of Flt3L-expanded NKDC to stimulate allogeneic T cells may result from their higher expression of costimulatory molecules (Fig. 4) , as it is known that more mature DC cause greater T cell stimulation. Previously, we have shown that NKDC stimulate CD4+ T cells to make significant amounts of IL-2 and minimal IFN-{gamma} in vitro (1) . CD8+ T cells, however, produce high levels of IFN-{gamma}, but negligible amounts of IL-2, after stimulation by NKDC. Their slightly increased antigen uptake and processing ability (Fig. 5) may also contribute to their increased function. Flt3L has a similar effect on DC, which after expansion become more phenotypically mature and functionally active (2 ,3 ,7) . The increased function of expanded NKDC could not be explained by their cytokine profile, as it was essentially unchanged (Fig. 6) .

The ability of NKDC to act as professional antigen-presenting cells is further established by their ability to cross-present antigen in vivo. Cross-presentation, which involves the uptake and presentation of exogenous antigens in the context of MHC I, is a process thought to be performed primarily by DC (32 33 34 35 36) . This phenomenon probably plays a critical role in stimulating CD8+ T cell responses to tumor and virus-infected cells (37 ,38) . Cross-presentation is also an attractive strategy for vaccinations aimed at raising CD8+ T cell responses. den Haan et al. (39 40 41) , as well as other groups, have shown that CD8{alpha}+, but not CD8{alpha}–, DC are responsible for cross-priming cytotoxic T cells in vivo. Previously, we have shown that NKDC are able to cross-present antigen in vitro, although at approximately one-third the concentration of conventional DC (1) . Our current data show that NKDC (which are CD8{alpha}low/- and remain so after Flt3L expansion, not shown) are also sufficient to cross-present exogenous antigen and induce cell division of antigen-specific CD8+ T cells in vivo (Fig. 7A ). As observed with in vitro T cell stimulation assays, Flt3L-expanded splenic NKDC perform this critical function more efficiently than normal NKDC, albeit at one-half the potency of conventional DC (Fig. 7B ), as measured by the number of CFSE+ T cells isolated from popliteal nodes. In vitro, FACS-purified NKDC and conventional DC loaded with ovalbumin actually induced similar proliferation of OT-I T cells (data not shown).

The exact origin of NKDC remains unknown, but there are data suggesting that NK cells and DC may share a common lineage. Marquez et al. (42) identified a bipotential NK cell and DC precursor in the human thymus. Miller et al. (43) described that a single adult human CD34+Lin–CD38+ progenitor cell can give rise to NK cells, B-lineage cells, DC, and myeloid cells. Meanwhile, murine fetal thymic progenitors have the potential to become T cells, NK cells, or DC, and in particular, NK cells and DC diverge from a common precursor (44) . Further evidence of a developmental relationship between NK cells and DC is suggested by our finding that treatment with Flt3L expands NKDC in addition to DC and NK cells (2 ,3 ,8) . In contrast, GM-CSF overexpression in vivo, as we have performed previously (1) , only expands DC (4 5 6) , but not NKDC (data not shown). It also remains unanswered whether NKDC are able to regulate DC function or possess the capacity to lyse immature or mature DC. Previously, Wilson et al. observed that immature DC were more susceptible than mature DC to lysis by autologous NK cells (45) .

Although NKDC have several features that are desirable for therapeutic manipulation, they have received little attention or have been largely ignored. In 1997, Josien et al. (46) first reported that most freshly isolated rat splenic DC express low levels of NK cell receptor protein-1 and that this marker becomes markedly up-regulated after overnight culture. In 2000, the same group published that CD4– DC, which constitute the majority of DC in the rat spleen, lyse Yac-1 targets (47) . In 2002, Homann et al. (48) reported that a population of DX5+CD11c+ splenocytes was increased in mice after infection with lymphocytic choriomeningitis virus (LCMV). In vitro, DX5+CD11c+ cells from LCMV-treated mice had both lytic and antigen-presenting function, and made IFN-{gamma}. In the presence of CD40 ligand blockade, however, adoptive transfer of these bitypic cells prevented autoimmune diabetes in transgenic mice expressing LCMV proteins in pancreatic islet cells, raising the possibility that, under certain conditions, NKDC may possess regulatory function, especially since they are immature at rest. Lian et al. (49) , in 2003, identified CD11c+NK1.1+ cells in the liver, spleen, lymph nodes, bone marrow, thymus, and blood of B6 mice. The group excluded these cells from their DC preparation to avoid confounding of phenotypic and functional analysis by NK or NKT cell contamination and did not study them further. There have been three very recent reports related to NKDC. Hanna et al. (50) reported that activated human NK cells can acquire costimulatory molecules and antigen-presenting capacity. Schleicher et al. (31) showed that NKDC-like cells in mice account for IFN-{gamma} production in macrophage preparations. Lastly, a study by Kamath et al. (51) demonstrated that DX5+NK1.1+CD11c+ splenocytes are responsible for all LPS-induced IFN-{gamma} production. However, the group concluded that these cells were NK cells, since they did not detect MHC II expression and did not study their antigen-presenting function. In contrast, we found that NKDC (defined as NK1.1+CD11c+ but which also express DX5) from normal mice express MHC II, as well as CD80 and CD86, albeit at low levels (Fig. 4) , and possess the capability of stimulating T cells to proliferate (Figs. 3B-D , 7 ). These findings make NKDC distinct from NK cells.

In summary, in vivo overexpression of Flt3L expands the numbers of functionally mature NKDC in lymphoid and nonlymphoid organs. These expanded NKDC retain the unique ability to lyse tumor cells and become more potent activators of naïve T cells, both in vivo and in vitro. Their increased ability to cross-present antigen suggests that Flt3L-expanded NKDC may be useful for immunotherapy. Our findings will facilitate the further study of these unique cells.

ACKNOWLEDGMENTS

This work was supported by NIH R01 Grant DK-068346. The authors have no conflict of interest.

Received for publication November 16, 2005. Accepted for publication December 28, 2005.

REFERENCES

  1. Pillarisetty, V. G., Katz, S. C., Bleier, J. I., Shah, A. B., Dematteo, R. P. (2005) Natural killer dendritic cells have both antigen presenting and lytic function and in response to CpG produce IFN-gamma via autocrine IL-12. J. Immunol. 174,2612-2618[Abstract/Free Full Text]
  2. Maraskovsky, E., Brasel, K., Teepe, M., Roux, E. R., Lyman, S. D., Shortman, K., McKenna, H. J. (1996) Dramatic increase in the numbers of functionally mature dendritic cells in Flt3 ligand-treated mice: multiple dendritic cell subpopulations identified. J. Exp. Med. 184,1953-1962[Abstract/Free Full Text]
  3. Miller, G., Pillarisetty, V. G., Shah, A. B., Lahrs, S., DeMatteo, R. P. (2003) Murine Flt3 ligand expands distinct dendritic cells with both tolerogenic and immunogenic properties. J. Immunol. 170,3554-3564[Abstract/Free Full Text]
  4. Pillarisetty, V. G., Miller, G., Shah, A. B., DeMatteo, R. P. (2003) GM-CSF expands dendritic cells and their progenitors in mouse liver. Hepatology 37,641-652[CrossRef][Medline]
  5. Hanada, K., Tsunoda, R., Hamada, H. (1996) GM-CSF-induced in vivo expansion of splenic dendritic cells and their strong costimulation activity. J. Leukoc. Biol. 60,181-190[Abstract]
  6. Miller, G., Pillarisetty, V. G., Shah, A. B., Lahrs, S., Xing, Z., DeMatteo, R. P. (2002) Endogenous granulocyte-macrophage colony-stimulating factor overexpression in vivo results in the long-term recruitment of a distinct dendritic cell population with enhanced immunostimulatory function. J. Immunol. 169,2875-2885[Abstract/Free Full Text]
  7. Shurin, M. R., Pandharipande, P. P., Zorina, T. D., Haluszczak, C., Subbotin, V. M., Hunter, O., Brumfield, A., Storkus, W. J., Maraskovsky, E., Lotze, M. T. (1997) FLT3 ligand induces the generation of functionally active dendritic cells in mice. Cell. Immunol. 179,174-184[CrossRef][Medline]
  8. Shaw, S. G., Maung, A., Steptoe, R. J., Thomson, A. W., Vujanovic, N. L. (1998) Expansion of functional NK cells in multiple tissue compartments of mice treated with Flt3-ligand: implications for anti-cancer and anti-viral therapy. J. Immunol. 161,2817-2824[Abstract/Free Full Text]
  9. Chakravarty, P. K., Alfieri, A., Thomas, E. K., Beri, V., Tanaka, K. E., Vikram, B., Guha, C. (1999) Flt3-ligand administration after radiation therapy prolongs survival in a murine model of metastatic lung cancer. Cancer Res. 59,6028-6032[Abstract/Free Full Text]
  10. Esche, C., Subbotin, V. M., Maliszewski, C., Lotze, M. T., Shurin, M. R. (1998) FLT3 ligand administration inhibits tumor growth in murine melanoma and lymphoma. Cancer Res. 58,380-383[Abstract/Free Full Text]
  11. Lynch, D. H., Andreasen, A., Maraskovsky, E., Whitmore, J., Miller, R. E., Schuh, J. C. (1997) Flt3 ligand induces tumor regression and antitumor immune responses in vivo. Nat. Med. 3,625-631[CrossRef][Medline]
  12. Peron, J. M., Esche, C., Subbotin, V. M., Maliszewski, C., Lotze, M. T., Shurin, M. R. (1998) FLT3-ligand administration inhibits liver metastases: role of NK cells. J. Immunol. 161,6164-6170[Abstract/Free Full Text]
  13. Viney, J. L., Mowat, A. M., O’Malley, J. M., Williamson, E., Fanger, N. (1998) Expanding dendritic cells in vivo enhances the induction of oral tolerance. J. Immunol. 160,5815-5825[Abstract/Free Full Text]
  14. Eto, M., Hackstein, H., Kaneko, K., Nomoto, K., Thomson, A. W. (2002) Promotion of skin graft tolerance across MHC barriers by mobilization of dendritic cells in donor hemopoietic cell infusions. J. Immunol. 169,2390-2396[Abstract/Free Full Text]
  15. Pillarisetty, V. G., Shah, A. B., Miller, G., Bleier, J. I., DeMatteo, R. P. (2004) Liver dendritic cells are less immunogenic than spleen dendritic cells because of differences in subtype composition. J. Immunol. 172,1009-1017[Abstract/Free Full Text]
  16. Carbone, F. R., Sterry, S. J., Butler, J., Rodda, S., Moore, M. W. (1992) T cell receptor alpha-chain pairing determines the specificity of residue 262 within the Kb-restricted, ovalbumin257–264 determinant. Int. Immunol. 4,861-867[Abstract/Free Full Text]
  17. Barnden, M. J., Allison, J., Heath, W. R., Carbone, F. R. (1998) Defective TCR expression in transgenic mice constructed using cDNA-based alpha- and beta-chain genes under the control of heterologous regulatory elements. Immunol. Cell Biol. 76,34-40[CrossRef][Medline]
  18. Kaisho, T., Akira, S. (2003) Regulation of dendritic cell function through toll-like receptors. Curr. Mol. Med. 3,759-771[CrossRef][Medline]
  19. Kaisho, T., Akira, S. (2001) Toll-like receptors and their signaling mechanism in innate immunity. Acta Odontol. Scand. 59,124-130[CrossRef][Medline]
  20. Sivori, S., Falco, M., Della Chiesa, M., Carlomagno, S., Vitale, M., Moretta, L., Moretta, A. (2004) CpG and double-stranded RNA trigger human NK cells by Toll-like receptors: induction of cytokine release and cytotoxicity against tumors and dendritic cells. Proc. Natl. Acad. Sci. U.S.A. 101,10,116-10,121[Abstract/Free Full Text]
  21. Granucci, F., Vizzardelli, C., Pavelka, N., Feau, S., Persico, M., Virzi, E., Rescigno, M., Moro, G., Ricciardi-Castagnoli, P. (2001) Inducible IL-2 production by dendritic cells revealed by global gene expression analysis. Nat. Immunol. 2,882-888[CrossRef][Medline]
  22. Granucci, F., Zanoni, I., Pavelka, N., Van Dommelen, S. L., Andoniou, C. E., Belardelli, F., Degli Esposti, M., Ricciardi-Castagnoli, P. (2004) A contribution of mouse dendritic cell-derived IL-2 for NK cell activation. J. Exp. Med. 200,287-295[Abstract/Free Full Text]
  23. Frucht, D. M., Fukao, T., Bogdan, C., Schindler, H., O’Shea, J. J., Koyasu, S. (2001) IFN-gamma production by antigen-presenting cells: mechanisms emerge. Trends Immunol. 22,556-560[CrossRef][Medline]
  24. Ohteki, T., Fukao, T., Suzue, K., Maki, C., Ito, M., Nakamura, M., Koyasu, S. (1999) IL 12-dependent interferon gamma production by CD8alpha+ lymphoid dendritic cells. J. Exp. Med. 189,1981-1986[Abstract/Free Full Text]
  25. Fukao, T., Matsuda, S., Koyasu, S. (2000) Synergistic effects of IL-4 and IL-18 on IL-12-dependent IFN-gamma production by dendritic cells. J. Immunol. 164,64-71[Abstract/Free Full Text]
  26. Hochrein, H., Shortman, K., Vremec, D., Scott, B., Hertzog, P., O’Keeffe, M. (2001) Differential production of IL-12, IFN-alpha, and IFN-gamma by mouse dendritic cell subsets. J. Immunol. 166,5448-5455[Abstract/Free Full Text]
  27. Stober, D., Schirmbeck, R., Reimann, J. (2001) IL-12/interleukin-18-dependent IFN-gamma release by murine dendritic cells. J. Immunol. 167,957-965[Abstract/Free Full Text]
  28. Munder, M., Mallo, M., Eichmann, K., Modolell, M. (1998) Murine macrophages secrete interferon gamma upon combined stimulation with IL-12 and IL-18: A novel pathway of autocrine macrophage activation. J. Exp. Med. 187,2103-2108[Abstract/Free Full Text]
  29. Puddu, P., Fantuzzi, L., Borghi, P., Varano, B., Rainaldi, G., Guillemard, E., Malorni, W., Nicaise, P., Wolf, S. F., Belardelli, F., Gessani, S. (1997) IL-12 induces IFN-gamma expression and secretion in mouse peritoneal macrophages. J. Immunol. 159,3490-3497[Abstract]
  30. Schindler, H., Lutz, M. B., Rollinghoff, M., Bogdan, C. (2001) The production of IFN-gamma by IL-12/interleukin-18-activated macrophages requires STAT4 signaling and is inhibited by IL-4. J. Immunol. 166,3075-3082[Abstract/Free Full Text]
  31. Schleicher, U., Hesse, A., Bogdan, C. (2005) Minute numbers of contaminant CD8+ T cells or CD11b+CD11c+ NK cells are the source of IFN-{gamma} in IL-12/interleukin-18-stimulated mouse macrophage populations. Blood 105,1319-1328[Abstract/Free Full Text]
  32. Albert, M. L., Pearce, S. F., Francisco, L. M., Sauter, B., Roy, P., Silverstein, R. L., Bhardwaj, N. (1998) Immature dendritic cells phagocytose apoptotic cells via alphavbeta5 and CD36, and cross-present antigens to cytotoxic T lymphocytes. J. Exp. Med. 188,1359-1368[Abstract/Free Full Text]
  33. Albert, M. L., Sauter, B., Bhardwaj, N. (1998) Dendritic cells acquire antigen from apoptotic cells and induce class I-restricted CTLs. Nature 392,86-89[CrossRef][Medline]
  34. Heath, W. R., Belz, G. T., Behrens, G. M., Smith, C. M., Forehan, S. P., Parish, I., Davey, G. M., Wilson, N. S., Carbone, F. R., Villadangos, J. (2004) Cross-presentation, dendritic cell subsets, and the generation of immunity to cellular antigens. Immunol. Rev. 199,9-26[CrossRef][Medline]
  35. Belz, G. T., Carbone, F. R., Heath, W. R. (2002) Cross-presentation of antigens by dendritic cells. Crit. Rev. Immunol. 22,439-448[Medline]
  36. Wilson, N. S., Villadangos, J. (2005) Regulation of antigen presentation and cross-presentation in the dendritic cell network: facts, hypothesis, and immunological implications. Adv. Immunol. 86,241-305[CrossRef][Medline]
  37. Melief, C. J. (2003) Mini-review: Regulation of cytotoxic T lymphocyte responses by dendritic cells: peaceful coexistence of cross-priming and direct priming?. Eur. J. Immunol. 33,2645-2654[CrossRef][Medline]
  38. Heath, W. R., Carbone, F. R. (2001) Cross-presentation in viral immunity and self-tolerance. Nat. Rev. Immunol. 1,126-134[CrossRef][Medline]
  39. den Haan, J. M., Lehar, S. M., Bevan, M. J. (2000) CD8(+) but not CD8(-) dendritic cells cross-prime cytotoxic T cells in vivo. J. Exp. Med. 192,1685-1696[Abstract/Free Full Text]
  40. Pooley, J. L., Heath, W. R., Shortman, K. (2001) Cutting edge: intravenous soluble antigen is presented to CD4 T cells by CD8- dendritic cells, but cross-presented to CD8 T cells by CD8+ dendritic cells. J. Immunol. 166,5327-5330[Abstract/Free Full Text]
  41. Iyoda, T., Shimoyama, S., Liu, K., Omatsu, Y., Akiyama, Y., Maeda, Y., Takahara, K., Steinman, R. M., Inaba, K. (2002) The CD8+ dendritic cell subset selectively endocytoses dying cells in culture and in vivo. J. Exp. Med. 195,1289-1302[Abstract/Free Full Text]
  42. Marquez, C., Trigueros, C., Franco, J. M., Ramiro, A. R., Carrasco, Y. R., Lopez-Botet, M., Toribio, M. L. (1998) Identification of a common developmental pathway for thymic natural killer cells and dendritic cells. Blood 91,2760-2771[Abstract/Free Full Text]
  43. Miller, J. S., McCullar, V., Punzel, M., Lemischka, I. R., Moore, K. (1999) Single adult human CD34(+)/Lin-/CD38(–) progenitors give rise to natural killer cells, B-lineage cells, dendritic cells, and myeloid cells. Blood 93,96-106[Abstract/Free Full Text]
  44. Shen, H. Q., Lu, M., Ikawa, T., Masuda, K., Ohmura, K., Minato, N., Katsura, Y., Kawamoto, H. (2003) T/NK bipotent progenitors in the thymus retain the potential to generate dendritic cells. J. Immunol. 171,3401-3406[Abstract/Free Full Text]
  45. Wilson, J. L., Heffler, L. C., Charo, J., Scheynius, A., Bejarano, M. T., Ljunggren, H. G. (1999) Targeting of human dendritic cells by autologous NK cells. J. Immunol. 163,6365-6370[Abstract/Free Full Text]
  46. Josien, R., Heslan, M., Soulillou, J. P., Cuturi, M. C. (1997) Rat spleen dendritic cells express natural killer cell receptor protein 1 (NKR-P1) and have cytotoxic activity to select targets via a Ca2+-dependent mechanism. J. Exp. Med. 186,467-472[Abstract/Free Full Text]
  47. Trinite, B., Voisine, C., Yagita, H., Josien, R. (2000) A subset of cytolytic dendritic cells in rat. J. Immunol. 165,4202-4208[Abstract/Free Full Text]
  48. Homann, D., Jahreis, A., Wolfe, T., Hughes, A., Coon, B., van Stipdonk, M. J., Prilliman, K. R., Schoenberger, S. P., von Herrath, M. G. (2002) CD40L blockade prevents autoimmune diabetes by induction of bitypic NK/DC regulatory cells. Immunity 16,403-415[CrossRef][Medline]
  49. Lian, Z. X., Okada, T., He, X. S., Kita, H., Liu, Y. J., Ansari, A., Kikuchi, K., Ikehara, S., Gershwin, M. E. (2003) Heterogeneity of dendritic cells in the mouse liver: identification and characterization of four distinct populations. J. Immunol. 170,2323-2330[Abstract/Free Full Text]
  50. Hanna, J., Gonen-Gross, T., Fitchett, J., Rowe, T., Daniels, M., Arnon, T. I., Gazit, R., Joseph, A., Schjetne, K. W., Steinle, A., Porgador, A., Mevorach, D., Goldman-Wohl, D., Yagel, S., LaBarre, M. J., Buckner, J. H., Mandelboim, O. (2004) Novel APC-like properties of human NK cells directly regulate T cell activation. J. Clin. Invest. 114,1612-1623[CrossRef][Medline]
  51. Kamath, A. T., Sheasby, C. E., Tough, D. F. (2005) Dendritic cells and NK cells stimulate bystander T cell activation in response to TLR agonists through secretion of IFN-alpha beta and IFN-gamma. J. Immunol. 174,767-776[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
U. I. Chaudhry, G. Plitas, B. M. Burt, T. P. Kingham, J. R. Raab, and R. P. DeMatteo
NK Dendritic Cells Expanded in IL-15 Exhibit Antitumor Responses In Vivo
J. Immunol., October 1, 2007; 179(7): 4654 - 4660.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
G. Plitas, U. I. Chaudhry, T. P. Kingham, J. R. Raab, and R. P. DeMatteo
NK Dendritic Cells Are Innate Immune Responders to Listeria monocytogenes Infection
J. Immunol., April 1, 2007; 178(7): 4411 - 4416.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
R. Yadav, D. J. Zammit, L. Lefrancois, and A. T. Vella
Effects of LPS-mediated bystander activation in the innate immune system
J. Leukoc. Biol., December 1, 2006; 80(6): 1251 - 1261.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
U. I. Chaudhry, T. P. Kingham, G. Plitas, S. C. Katz, J. R. Raab, and R. P. DeMatteo
Combined Stimulation with Interleukin-18 and CpG Induces Murine Natural Killer Dendritic Cells to Produce IFN-{gamma} and Inhibit Tumor Growth
Cancer Res., November 1, 2006; 66(21): 10497 - 10504.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Summary
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
fj.05-5411fjev1
fj.05-5411fjev2
20/7/982    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chaudhry, U. I.
Right arrow Articles by DeMatteo, R. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chaudhry, U. I.
Right arrow Articles by DeMatteo, R. P.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS