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(Journal of Leukocyte Biology. 2002;71:941-949.)
© 2002 by Society for Leukocyte Biology

Flt-3 ligand (FL) drives differentiation of rat bone marrow-derived dendritic cells expressing OX62 and/or CD161 (NKR-P1)

Cynthia S. Brissette-Storkus*,{dagger}, J. C. Kettel{ddagger}, T. F. Whitham{dagger},§, K. M. Giezeman-Smits{dagger}, L. A. Villa{dagger},||, D. M. Potter# and William H. Chambers{dagger},||

* Eye and Ear Institute and Department of Ophthalmology,
{dagger} Brain Tumor Center of the University of Pittsburgh Cancer Institute, and the Departments of
{ddagger} Molecular Genetics and Biochemistry,
§ Neurological Surgery,
|| Pathology, and
# Biostatistics, University of Pittsburgh School of Medicine, Pennsylvania

Correspondence: Cynthia S. Brissette-Storkus, 919 Eye and Ear Institute, University of Pittsburgh, 203 Lothrop Street, Pittsburgh, PA 15213. E-mail: cbstork{at}pitt.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bone marrow-derived dendritic cells (DC) of the rat have not been as well characterized as those from the mouse. Here, large quantities of bone marrow-derived rat DC were generated when Flt-3 ligand (FL) was used as an adjunct to granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4). These cells displayed a typical DC phenotype, expressing MHC class II, CD54, CD80, CD86, and CD11b/c. These DC also uniformly expressed low levels of CD161 and expressed OX62 in a bimodal distribution. Few cells were recovered from cultures grown without FL, and they failed to express OX62 or CD161. The DC generated with FL were more potent antigen-presenting cells in mixed lymphocyte cultures than cells grown without FL, and among FL-derived cells, the OX62+ cells were slightly more stimulatory than OX62- cells. Thus, FL is a useful cytokine for obtaining large quantities of functional rat DC subsets in vitro.

Key Words: IL-4 • GM-CSF • integrin • MHC • MLR


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dendritic cells (DC) exist in vivo as highly potent, but rare antigen-presenting cells, typically comprising <=1% of the mononuclear cell population of various lymphoid organs [1 ]. Recent advances in the development of techniques for expanding DC in vitro have done much to further the study of basic DC biology. Importantly, the ability to generate large quantities of DC has also made it feasible to develop DC-based vaccines for use in animal models and a variety of clinical trials. The rat is a frequently used animal for many disease models. However, studies on the in vitro generation and phenotyping of DC from rat bone marrow (BM) cultures lag far behind those performed in the mouse or human systems. Techniques used to generate DC from rat BM have included culturing without exogenous cytokines, the addition of conditioned mediums, and the use of granulocyte macrophage-colony stimulating factor (GM-CSF) in combination with gelatin-coated flasks or the cytokine interleukin-4 (IL-4) [2 3 4 5 6 ]. In most cases, the numbers of DC obtained in vitro have been disappointingly low, typically being ~5% or less of the input cell number. With the combination of GM-CSF plus IL-4, greater numbers of rat DC have been generated, but the yield was still only 30–40% of the input cell number [6 ].

Phenotyping cultured rat DC has been hampered in part by a paucity of reagents that detect rat DC-specific epitopes. However, the recently identified marker, OX62, has proved useful for defining certain populations of rat DC. Anti-OX62 was derived against veiled DC of rat thoracic lymph, and this antibody recognizes the rat homologue of integrin-{alpha}E2 [7 , 8 ]. The expression of OX62 on major histocompatibility complex (MHC) class II+ cells is restricted to DC from the lymph, spleen, thymus, nodes, and various nonlymphoid organs [7 ]. The OX62 antibody also reacts with a population of MHC class II- dendritic epidermal {gamma}{delta} T cells, although these can be discriminated easily from DC based on their expression of T cell receptor components and lack of MHC class II [8 ]. The OX62 epitope does not appear to be expressed on all DC, as Langerhans cells in the skin do not react with this reagent [7 ]. At present, it is not known whether the expression of OX62 in vivo might be restricted to a particular subset of DC (i.e., myeloid vs. lymphoid or plasmacytoid) or is expressed differentially during activation/maturation of DC. It is interesting that OX62 is expressed variably on in vitro-generated DC. Among in vitro-generated DC, it was observed that a proportion of the DC generated with GM-CSF and gelatin-coated flasks expressed OX62 [5 ]. In contrast, the DC obtained from cultures with GM-CSF and IL-4 did not express OX62 [6 ]. Thus, the generation of DC that express OX62 in vitro may be well regulated by culture conditions.

Here, we have examined the phenotype of rat BM-derived DC generated in the presence of flt-3 ligand (FL), which is a cytokine known to stimulate the proliferation of hematopoietic progenitor and stem cells [9 , 10 ]. Administration of FL to animals in vivo has been shown to cause a massive increase in the number of mature DC and DC progenitors found systemically in various organs [11 12 13 ]. A limited number of studies have also demonstrated that FL has an enhancing effect on the numbers of DC generated in vitro as well [14 , 15 ]. This ability of FL to boost DC yields has made it an attractive cytokine for application to in vitro cultures of rat BM-derived DC. We found that when FL was added to BM cultures containing GM-CSF and IL-4, there was an approximate sevenfold increase in the yield of DC generated. Further, a large percentage of these cells coexpressed OX62 and CD161 [natural killer cell receptor protein 1 (NKR-P1)]. CD161, a C-type lectin best known for being a signaling molecule on NK cells [16 17 18 ], has been described recently on DC as well [19 , 20 ]. In the absence of FL, neither OX62 nor CD161 was expressed on the BM-derived cells. Thus, we demonstrate that FL has at least two effects on rat BM-derived DC. First, it increases the number of DC generated; second, it appears to play a significant role in modulating the phenotype of the resulting DC populations. Our data further provided the first description of CD161 expression on rodent DC generated in vitro.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Fischer F344 (F344) male rats weighing 75–125 g were purchased from Taconic Farms (Germantown, NY). A-strain Copenhagen Irish (ACI) male rats weighing 110–135 g were purchased from Harlan Sprague Dawley (Indianapolis, IN). Animals were maintained in a specific, pathogen-free facility at the University of Pittsburgh (PA) and were used under an IACUC-approved protocol.

Reagents, cytokines, and antibodies
RPMI-1640 nonessential amino acids, HEPES buffer, and sodium pyruvate were purchased from Mediatech (Herndon, VA). Penicillin/streptomycin was purchased from Gibco-Life Technologies, Inc. (Grand Island, NY). Fetal bovine serum (FBS) was purchased from BioWhittaker (Walkersville, MD). ß Mercaptoethanol (2-ME) was purchased from Sigma Chemical Co. (St. Louis, MO). NG-monomethyl-L-arginine (NMA) was purchased from Calbiochem (La Jolla, CA). Ficoll-Paque was purchased from Pharmacia Fine Chemicals (Piscataway, NJ). Recombinant mouse GM-CSF and IL-4 were a gift of Schering-Plough (Kenilworth, NJ). Recombinant mouse FL was purchased from R&D Systems (Minneapolis, MN). Unconjugated and/or fluorochrome-labeled monoclonal antibody (mAb) OX62 (anti-integrin {alpha}E), W3/25 (anti-CD4), OX8 (anti-CD8), OX39 (anti-IL-2r {alpha}), and OX33 (anti-B cells) were purchased from Serotec (Oxford, UK). Unconjugated and/or fluorochrome-labeled mAb OX42 (anti-CDllb/c), OX6 (anti-MHC class II), 3H5 (anti-CD80 [B7.1]), 2F4 (anti-CD86 [B7.2]), G4.18 (anti-CD3), and streptavidin-phycoerythrin were purchased from Pharmingen (San Diego, CA). Cy-5-conjugated fragments of mAb 3.2.3 (anti-CD161 [NKR-P1]) were prepared as described previously by us [16 ]. Biotinylated 1A29 (anti-CD54 [intercellular adhesion molecule-1]) and fluorescein isothiocyanate (FITC)-conjugated F(ab')2 fragments of goat anti-mouse immunoglobulin G (IgG) were purchased from Caltag (Burlingham, CA). Cy-3-conjugated goat anti-mouse IgG was purchased from Jackson Laboratories (West Grove, PA).

BM cultures
The femurs and tibias from the hind limbs of F344 rats were removed, cleaned of muscle tissue, and placed in sterile petri dishes (Falcon, Franklin Lakes, NJ) containing complete medium [RPMI 1640 with 5% FBS, pen/strep (100 U/ml and 100 ug/ml), and 2 mM L-glutamine]. The ends of the bones were cut, and the marrow was flushed out with culture medium (CM) in a 10-cc syringe (Becton-Dickinson, Franklin Lakes, NJ) capped with a 23-gauge needle (Becton-Dickinson). The marrow was dispersed and filtered through a sterile, nylon screen (Falcon) to remove debris and clumps. The cells were pelleted at 400 g for 5 min, and red blood cells in the pellet were lysed by hypotonic treatment with 17 mM Tris/14 mM NH4Cl, pH 7.2, for 2 min. Isotonicity was restored by the addition of 10x volume of RPMI 1640/10% FBS. After pelleting, the BM cells were resuspended in DC CM [DCM; RPMI 1640, 10% FBS, pen/strep (100 U/ml and 100 µg/ml), 2 mM glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 50 µM HEPES buffer, 5 x 10-5 M 2-ME, and 0.5 mM NMA]. NMA is a nitric oxide (NO) synthase inhibitor that prevents NO-mediated suppression of rat lymphocyte proliferation in vitro [21 , 22 ]. The DCM was supplemented further with 500 U/ml recombinant (r)GM-CSF, 500 U/ml rIL-4, and for some cultures, 25 or 50 ng/ml FL. Cells (3 ml) were then plated into six-well trays (Costar, Corning, NY) at 6–9 x 106 cells/well and were maintained in a humidified incubator at 37°C with 5% CO2/95% air. The cultures were fed every other day by removing 50% of the supernatant and replacing it with fresh DCM plus cytokines. On day 4, the plates were gently swirled, taking care not to disrupt any loose clusters of cells, and the nonadherent cells and medium were removed. Fresh DCM (2 ml) plus cytokines were added immediately to each well. The spent medium was centrifuged to pellet the contaminating cells, and 2 ml/well old medium was added back into the trays. This procedure removes the majority of contaminating granulocytes and lymphocytes from the BM preparations. On day 8, the nonadherent cells were aspirated, the wells were washed once with medium, and the cells were pooled. DC cultures that were matured for an additional day were placed in fresh DCM containing rGM-CSF and rIL-4 and cultured in a new six-well tray.

Flow cytometric analysis and cell sorting
Cell populations were stained in suspension with the indicated antibodies for single-, double-, or triple-color flow cytometric analysis. Analyses on a minimum of 3500-gated events were conducted on a Becton-Dickinson FACStar Plus, FACStar, or FACSan flow cytometer at the Flow Cytometry Facility of the Pittsburgh Cancer Institute (PA). Data were analyzed, and graphics of the collected data were generated using the Repro ManTM program (True Facts Software Inc., Seattle, WA). Sorting live cells was performed on a FACStar Plus as described previously [16 , 23 ].

Cytospin slide preparation
BM-derived cells were adjusted to a concentration of 2–4 x 105 cells/ml in RPMI 1640, and 200 µl cell suspension was spun onto a slide at 200 rpm for 5 min in a Shandon Cytospin 2. The slides were air dried and then fixed in methanol for 5 min. After this, the slides were air dried again and then stained using Accustain (Sigma Chemical Co.), a modified Giemsa stain. Slides were placed in Accustain for 2 min, rinsed briefly in deionized water to remove excess stain, air dried, and then examined microscopically.

Mixed leukocyte reaction
The nylon wool, nonadherent fraction of splenic lymphocytes of ACI rats (RT1a) was used as a responder cell. Stimulator cells were BM-derived DC or splenic mononuclear cells of F344 rats (RT11v1). To obtain mononuclear cells, spleens of rats were asceptically removed and minced to yield a single cell suspension. Mononuclear cells were then purified by centrifugation on Ficoll-Paque, and the nylon wool, nonadherent cells were isolated as described previously [16 ]. One-way mixed leukocyte reactions were performed in U-bottom 96-well microtiter plates (Costar) in medium identical to DCM, with the exception of the HEPES buffer concentration, which was 10 mM. No cytokines were added. A constant number of responder cells (1x105/well) were mixed with graded numbers of {gamma}-irradiated (2000 rads) stimulator cells, and the plates were cultured for 4 days in a humidified incubator at 37°C with 5% CO2/95% air. During the final 18 h of culture, cells were labeled with 1.0 µCi/well [3H]-thymidine (specific activity, 2 Ci/mM; New England Nuclear, Beverly, MA). Plates were harvested with the Unifilter-96 automatic harvester (Packard, Downers Grove, IL). Thymidine incorporation was determined using the TopCount microplate scintillation counter (Packard). Statistics were performed using the Wilcoxon rank sum test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
FL influences the generation and phenotype of rat BM-derived DC
As FL has been shown to substantially increase the number of DC precursors in vivo [11 12 13 ], we evaluated whether the addition of FL to in vitro rat BM cultures would enhance the yield of rat DC. BM cells obtained from Fischer 344 rats were cultured with rGM-CSF and rIL-4, with or without the addition of FL to the cultures. In our system, the BM cells were fed every other day by replacing 50% of the spent medium with fresh medium and cytokines. Nonadherent cells were gently removed and discarded on day 4 of culture, and the cellular aggregates on the stromal layer were retained. On day 8 of culture, nonadherent and loosely adherent clusters of cells were recovered for analyses. In these experiments, we observed that adding FL to cultures enhanced the yield of recovered cells dramatically. The percentage of cells obtained with GM and IL-4 alone was never more than 10% of the input cell number. In contrast, cultures containing FL routinely resulted in recovery of 25–60% of the starting BM cell number, averaging a sevenfold increase over the number of cells recovered in the absence of FL (Table 1 ).


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  		Table 1. Flt-3 Ligand Dramatically Increases the Yield of Nonadherent Cells Recovered from Rat Bone-Marrow Dendritic Cell Cultures

 
Flow cytometric analyses were performed to characterize the phenotype of the BM-derived DC generated in the presence or absence of FL (Fig. 1 ). When stained with antibody against OX42 (CD11b/c), the majority of the cells grown in GM-CSF and IL-4 appeared to express low levels of this epitope because the mean fluorescence intensity (MFI) of this population as a whole is shifted compared with the control curve (MFI control=32; MFI OX42=97). Similar results were seen for CD4 (MFI control=32; MFI CD4=99). A subpopulation of cells, 55 ± 12% (n=3) expressed CD54, and approximately 70 ± 7% (n=4) expressed MHC class II. The cultured cells lacked the markers CD8{alpha}, CD3, and OX33 (pan B cell; not shown). Notably, cells from these cultures lacked expression of OX62 and CD161 (Fig. 1) . Morphologically, the cells grown in GM and IL-4 tended to be smaller than the cells recovered from cultures with FL, and most lacked obvious dendrites or veiling (not shown).



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  		Figure 1. Comparison of the phenotype of BM cultures grown for 8 days in the presence of GM-CSF and IL-4 (GM/4) versus GM-CSF, IL-4, and FL (GM/4/FL). FACS analysis for the indicated markers was performed with the appropriate anti-rat antibodies. Shaded area, specific Ab staining; open area, isotype-control Ab staining.

 
Phenotypic characterization of the cells grown with FL in addition to GM-CSF and IL-4 demonstrated that the majority of these cells, 72 ± 9% (n=2), also expressed OX42 (CD11b/c). The positive cells stained as a distinct peak, indicating a greater specific level of expression compared with cells grown only with GM-CSF and IL-4 (Fig. 1) . A small but discrete percentage of FL-treated cells (28±3%; n=5) was positive for CD4. When examined for the expression of MHC class II, the cells generated with FL exhibited three distinct subsets including MHC II- (19±12%), MHC IIlo (75±10%), and MHC IIhi (7±3%) cells (n=7). Evaluation of CD54 expression indicated that the majority of the cells were CD54lo (86±8%), and a minor subset was CD54hi (5±3%; n=5). As most of the cells expressed only low-to-intermediate levels of MHC class II or CD54, the data suggest that the majority of BM-derived cells were of a less mature DC phenotype. It is interesting that FL supported the generation of a large subset of cells (49±13%; n=8), expressing the epitope recognized by OX62. Cells obtained in the presence of FL also expressed a low level of CD161. In the representative figure shown, the MFI of the control Ab was 4.1, and specific staining with CD161 gave a MFI of 13.4. The FL-treated, BM-derived cells were negative for expression of CD8{alpha}, CD3, and OX33 (unpublished results). When examined microscopically, most cells in these cultures appeared large and irregular in shape, many had numerous short membrane processes, and a few had long dendrites, but most cells lacked extensive veiling. This type of physical morphology is also consistent with these cultures being primarily composed of immature DC.

Phenotypic maturation of BM-derived DC
The cells from DC cultures generated in the presence of FL were of particular interest because they expressed the DC marker OX62, and in contrast to most attempts to culture rat DC, large quantities of cells could be easily obtained. The initial phenotyping of these cells as described in Figure 1 demonstrated that the majority of the cells expressed low-to-intermediate levels of MHC class II and the adhesion/accessory molecule CD54. As functionally mature DC are known to express high levels of MHC class II, CD54, and other costimulator molecules including CD80 and CD86 [24 25 26 ], our data suggested that we had generated predominantly immature DC.

To determine if we could increase the proportion of cells with a mature DC phenotype, we subcultured the day 8 BM-derived cells that had been grown with GM-CSF, IL-4, and FL. Nonadherent cells were removed from their original culture plates and transferred to new wells with fresh medium containing GM-CSF and IL-4, but no FL. The surface expression of five antigens, MHC class II, CD54, CD80, CD86, and CD161, was then compared on day 8 and after 1 day of subculture (i.e., day 9) to determine if the additional culture resulted in an increase in the number of mature DC (Fig. 2 ). Notably, a large subpopulation of cells up-regulated their expression of MHC class II, CD54, or CD86 on day 9. The majority of the MHC class II+ cells present on day 8 existed in an MHC IIlo peak with only a few cells present in the MHC IIhi peak. However, the percentage of cells with bright surface expression of MHC class II increased from 7 ± 3% on day 8 to 60 ± 12% (n=7) on day 9. The MFI of the day 8 cells in the MHC IIlo population was 6- to 14-fold lower than the MFI of cells in the MHC IIhi peak (day 8 or 9). Similarly, on day 8, only 5 ± 3% of the cells were CD54hi, whereas 51 ± 10% (n=5) of the cells were CD54hi on day 9. The MFI of cells staining brightly for CD54 on day 9 was 5–14 times increased over the MFI of the CD54lo-positive cells present at days 8 and 9. Only a few of the DC, 13 ± 5% (n=3), expressed the costimulator molecule CD86 on day 8, but at day 9, 70 ± 8% (n=2) of the cells were CD86-positive. CD80 was also increased by day 9, although the results were less dramatic. The costimulator molecule CD80 was detected weakly on the day 8 cells (MFI=11), but on day 9, the relative staining intensity of the whole population (MFI=21) had increased approximately twofold. Similarly, in four of five experiments, the percentage of cells that stained positive for CD161 at day 9 was increased by 31 ± 15% compared with day 8. Two distinct populations of OX62+ and OX62- DC were still observed in the recovered day 9 cells, with no change in the intensity of staining or the proportion of cells expressing the marker (unpublished results). The yield of viable cells on day 9 was 55–75% of the input cells on day 8. The decrease in cell number from day 8 to day 9 is mostly due to firm adherence of a subset of cells to the culture well, while very little cell death was observed. These adherent cells could potentially represent immature DC or monocytic cells expressing low levels of MHC class II and accessory molecules. Selective depletion of these cells could partially contribute to the enrichment for mature DC obtained on day 9. However, the increase in the percentage of MHC IIhi cells recovered is still substantial enough to conclude that spontaneous maturation of a significant proportion of the immature DC population likely occurred between day 8 and day 9.



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  		Figure 2. Day 9 BM-derived cultures are enriched for mature DC. FACS analysis for the indicated cell-surface markers was performed on day 8 (d8) of cultures generated with GM-CSF, IL-4, and FL (top panels) and on day 9 (d9) after 18–24 h of subculture in the absence of FL (bottom panels). Shaded area, specific Ab staining; open area, isotype-control Ab staining.

 
In morphological appearance, the day 9 DC were also large and irregular in shape, but appeared to have more extensive membrane processes and veiling than did the day 8 cells (not shown). Cytospins of the day 9 mature cells confirmed the dendritic morphology of these cells (Fig. 3 ). Many cells displayed pronounced, veil-like cytoplasmic membrane processes. The nuclei of most of the cells were typically irregular and oblong-shaped, but numerous cells also displayed a twisted or cloverleaf-shaped nucleus similar to another study illustrating cultured rat DC [5 ].



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  		Figure 3. Giemsa-stained cytospin of day 9 mature DC derived from BM cells cultured with GM-CSF, IL-4, and FL; original, 40x objective.

 
Coexpression of CD161 with MHC class II and OX62
The DC generated in the presence of FL were of particular interest as they expressed the DC markers OX62 and CD161. The C-type lectin CD161 is most commonly found on NK cells and NK-T cells and has been described recently to be expressed at low levels on some DC. However, the expression of CD161 on in vitro-derived rat BM DC has not been described previously. We therefore carried out a more extensive characterization of this population of cells using two-color fluorescein-activated cell sorter (FACS) analyses. Coexpression of MHC class II and CD161 on days 8 and 9 DC is shown in Figure 4A and 4B . As described above, ~90% of immature day 8 DC expressed intermediate levels of MHC class II expression. However, these same cells also expressed low levels of CD161. A distinct positive peak of CD161-positive cells was not observed; rather, the whole cluster was shifted away from the baseline of negative-control staining (i.e., the horizontal line demarcating quadrants 3 and 4). As the percentage of mature DC in the recovered cells increased at day 9, the detection of CD161 became more pronounced. It can be seen that a large fraction of the mature MHC class II "bright" DC are clearly positive for CD161. The apparent increase in intensity of CD161 expression on day 9 MHC class II "bright" cells is two- to threefold that of the day 8 MHC class II "intermediate" cells. We also examined the distribution of CD161 and MHC class II on OX62+ DC. CD161 was expressed at comparable levels by OX62+/MHC class II+ cells and OX62-/MHC class II+ DC (Fig. 5A and 5B ). Thus, there did not appear to be differential expression of this marker on the various subsets of DC generated in culture.



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  		Figure 4. Coexpression of CD161 and MHC class II on DC derived from BM cultures grown with GM-CSF, IL-4, and FL. Dual-color FACS analysis of MHC class II expression (x-axis, FL1) versus CD161 expression (y-axis, FL4) was performed with FITC-conjugated mAb OX6 and Cy-5-conjugated mAb 3.2.3. Quadrant parameters were determined using the appropriate isotype controls. (A) Day 8 cells; (B) matured day 9 cells.

 


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  		Figure 5. CD161 expression on OX62+/MHC class II+ and OX62-/MHC class II+ DC. Three-color FACS analysis was performed on day 9 DC using FITC-conjugated mAb OX6 (x-axis, FL1), Cy-5-conjugated mAb 3.2.3 (y-axis, FL4), and mAb OX62 plus Cy-3-conjugated goat anti-mouse Ig. Gates were selected based on OX62 expression as compared with isotype controls and analysis of MHC class II versus CD161 shown for these gates. (A) Phenotype of OX62-negative cells; (B) phenotype of OX62-positive cells.

 
Stimulatory capacity of rat BM DC
The hallmark of functional DC is their ability to stimulate antigen-specific T cell responses. Therefore, to determine whether the cells in our cultures presented antigen efficiently, cells from day 9 DC cultures were used as stimulators in a primary allogeneic mixed lymphocyte reaction (MLR). Graded numbers of irradiated DC were added to 1 x 105 nylon wool, nonadherent T cell-enriched splenocytes from allogeneic ACI rats. For comparison, equivalent numbers of unseparated F344 splenocytes were also used as stimulators. Splenic leukocytes contain 1–2% DC [1 , 11 ], and it has been shown that approximately 80% of rat splenic DC express OX62 [27 ]. As shown in Figure 6A , the BM-derived DC were more than 100 times more potent at stimulating MLR proliferation than the unfractionated splenocytes, as the thymidine incorporation stimulated by 1 x 103 DC was four to five times that stimulated by 1 x 105 splenocytes. We also compared the stimulation capacity of day 9 mature DC harvested from FL-treated cultures with that of day 9 cells harvested from GM-CSF- and IL-4-only cultures, after also subculturing the latter for 1 day in fresh media with GM-CSF and IL-4. The DC from the FL-containing cultures were at least 10 times more potent at allostimulation than the cells grown with GM-CSF and IL-4 alone (Fig. 6B) . This was particularly evident at conditions of higher stimulator cell number, where the GM/IL-4-cultured cells appeared to become suppressive. We next sorted the FL-grown DC based on their expression of OX62 to determine if there was a functional difference that correlated with the expression of this marker. The results of two experiments are shown in Figure 7 . Overall, the OX62+ and OX62- DC were potent MLR-stimulator cells (Fig. 7A and 7B ). However, at lower cell numbers, the OX62+ DC appeared to have slightly enhanced stimulatory capacity. In one experiment (Fig. 7A) , at cell doses from 3 x 101–3 x 103 per well, the purified OX62+ DC were significantly stronger (P<.01; Wilcoxon rank sum test) at stimulating the MLR response than OX62- DC. In a second experiment (Fig. 7B) , the OX62+ DC stimulated better than the OX62- DC at cell numbers from 3 x 101–3 x 102 per well (P<.01). In an additional experiment with magnetically sorted DC (unpublished results), stimulation with OX62+ DC exceeded that of OX62- DC at 3 x 102–1 x 104 cells per well (P<.01). As it was possible that the OX62- population of cells contained immature DC or cells of a different lineage, we also determined by FACS (unpublished results) the percentage of cells that were strongly positive for CD86 expression within the OX62- and OX62+ subsets. In our hands, this marker correlates well with DC maturation (Fig. 2) . The percentage CD86 expression was used to calculate the number of CD86-positive cells in the populations and compare stimulation capacity (Fig. 7C and 7D ). A difference between the OX62- and OX62+ cells was still apparent, even when the results were adjusted for the number of mature DC within each population. Thus, the OX62+ DC were consistently more stimulatory in MLR than OX62- DC.



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  		Figure 6. Stimulation capacity of day 9 mature DC in allogeneic MLR. F344 rat BM-derived DC derived with GM-CSF, IL-4, and FL (•) were compared with (A) fresh F344 rat splenocytes ({blacksquare}) or (B) day 9 BM cells cultured with GM-CSF and IL-4 ({circ}) for their ability to stimulate allogenic ACI rat (1x105/well) nylon wool, nonadherent, T-cell-enriched splenocytes.

 


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  		Figure 7. Stimulation capacity of OX62+ versus OX62- DC. Mature, day 9 DC derived from cultures with GM-CSF, IL-4, and FL were stained with FITC-conjugated OX62 mAb and FACS sorted into purified OX62+ and OX62- populations (>97% purity). (A and B) Stimulation capacity of graded numbers of unsorted ({circ}), OX62- ({blacktriangledown}), and OX62+ (•) DC in two MLR experiments. (C and D) Stimulation capacity of the cells based on the calculated number of B7.2-positive cells in the corresponding OX62- ({circ}) versus OX62+ (•) populations.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have shown here that in the presence of FL, GM-CSF, and IL-4, large quantities of functional rat DC can be generated easily and consistently in vitro. These cells had a typical DC phenotype and were positive for the expression of MHC class II, CD54, CD80, CD86, and CD11b/c. It is interesting that a subset of these cells stained positive with OX62, a mAb specific for some rat DC. Our results differ from those shown for rat DC generated in the presence of GM-CSF and IL-4 alone, in which OX62 reactivity was not detected. The DC grown with FL also expressed the C-type lectin CD161. Recently, this molecule has been described as being expressed by freshly isolated rat DC [20 ], but its expression by in vitro-grown BM-derived DC was unknown prior to these studies. In our hands, CD161 was not expressed on cells grown in the absence of FL, although it is possible that other cytokine combinations may induce expression of this molecule.

An important feature of our in vitro culture system is that populations consisting of predominantly immature or predominantly mature DC can be generated depending on the conditions. This should prove to be a useful feature for the study of rat DC biology, as well as for designing rat DC vaccine models. As a result of DC maturation, which occurred between days 8 and 9, we observed up-regulation of the expression of CD54, CD80, CD86, and CD161 and particularly strong up-regulation of MHC class II on the cells. For MHC class II, this most likely represents a shift of preformed MHC class II molecules stored in intracellular lysosomes to the cell surface, because immature rat DC have been shown to have these organelles in abundance [6 ]. The exact trigger for this maturation process in our system is unknown. Up-regulation of MHC class II occurred when BM DC were removed from their adherent clusters and placed in fresh cultures in the absence of FL. It is possible that in our system, FL promotes maintenance of DC at an immature differentiation stage and that withdrawal from this cytokine allows rapid DC maturation. It has been shown that DC isolated from the livers of mice, administered FL in vivo, exhibited a predominantly immature phenotype and required ex vivo culturing in GM-CSF and IL-4 before the isolated cells acquired a mature phenotype and function [13 ]. It is also possible that the stromal cells provide an additional signal that prevents DC maturation, as it has been shown that immature DC derived in GM-CSF and IL-4 alone, when placed in fresh cultures without cytokine, mature within 2 days [6 ]. Alternatively, the adherent cells in these original cultures may be a perpetual source of developing immature precursor DC that otherwise "dilute" out nonadherent DC of a more mature phenotype.

A unique phenotype we observed in DC from cultures containing FL was the expression of OX62 and CD161. The exact function of the OX62 or CD161 molecules on rat DC is unknown. The molecule defined by OX62 has been identified as an integrin and is therefore likely to function in cell adhesion. Two similar but not identical integrin {alpha} subunits, E1 and E2, have been identified in the rat [8 ]. The rat {alpha} E1 subunit is virtually identical (99% sequence identity) with the mouse {alpha} E1 integrin, also known as {alpha} M290. The unique rat integrin {alpha} E2, defined by mAb OX62, also shows a high degree of homology (89% sequence identity) with mouse integrin {alpha} E1 subunit. Expression of mouse integrin {alpha} E1 has been associated with T cell populations that localize within epithelium, including dendritic epidermal {gamma}{delta} T cells [8 ] and intraepithelial lymphocytes, at virtually all mucosal epithelial sites [28 , 29 ]. Similarly, rat {alpha} E2, as detected with mAb OX62, is also expressed by dendritic epidermal {gamma}{delta} T cells and intraepithelial T cells in the intestine [8 ]. Functionally, mouse integrin {alpha} E1 has been shown to bind to E-cadherin expressed on epithelial cells [30 ]. Thus, it seems likely that differential expression of {alpha} E integrins may affect the migration and/or localization of cells that express these molecules.

Although rat integrin {alpha} E2 (OX62) is expressed on subpopulations of MHC class II+ DC in the lymph, lymph nodes, and spleen, it does not appear to be present on DC in the intestinal epithelium or on Langerhans cells in skin epidermis [7 ]. Similarly, mouse Langerhans cells from the skin do not express integrin {alpha} E1, but it can be detected on a subpopulation of DC in the lymph nodes [31 ]. Because Langerhans cells are thought to traffic to the lymph nodes after antigen stimulation [32 , 33 ], it is possible that in vivo, certain DC start to express the {alpha} integrins during their migration from the periphery to the lymph organs. This would account for the large percentage of DC expressing OX62 found in thoracic lymph. These integrins may then be involved in the microanatomical localization of DC within the lymph organs. Alternately, it is possible that the DC, which express these integrins, represent a distinct subset of DC that is perhaps derived from precursors that have been exposed to specific cytokines such as FL. This is supported by our data shown here, which demonstrate that OX62 was only expressed on DC grown in the presence of FL. Interestingly, we found that the OX62+ subset of BM-derived DC appeared to have a slightly better stimulation capacity in MLR than the OX62- cells. This suggests that this subset of DC may indeed have unique or heightened functional capabilities.

Our data are consistent with previous observations that rat DC isolated from spleen or thymus can express CD161, a molecule identified previously on NK cells and NK-T cells [16 , 17 ]. Our data demonstrate that CD161 can also be expressed specifically on rat BM-derived DC cultured in the presence of FL. It remains to be determined at the molecular level what form(s) of NKR-P1 DC express. In rodents, CD161 is known to be a multigene family. At least three similar genes have been described in mice and rats [17 , 34 35 36 ]. Differences in the form(s) of CD161 expressed by DC would be likely to confer functional consequences. The product of the NKR-P1A gene in rats and the NKR-P1C gene in mice is an activating molecule on NK cells and T cells, capable of stimulating cytotoxicity and cytokine release [17 , 34 35 36 37 38 ]. In contrast, the NKR-P1B gene of mice appears to deliver inhibitory signals to cells [37 ]. The mAb used in our studies, 3.2.3, is known to react with the rat NKR-P1A product and does not appear to react with the NKR-P1D product [17 , 40 ]. Reactivity of this mAb with the products of other NKR-P1 genes is unknown. Thus, it is likely that rat DC express at least one activating form of CD161. It is also possible that DC may express more than one form of CD161 simultaneously, as we have previously observed coexpression of multiple CD161 gene products in NK clones [40 ].

Little is known yet about the physiological function of CD161 molecules on DC, which have recently been shown to be capable of cytolytic activity against certain tumors [20 , 27 ], and it has been suggested that CD161 may be involved in such killing [20 ], particularly because cross-linking CD161 on NK cells and T cells triggers cytolytic function. However, there is no direct evidence to support a role for CD161 in DC cytolytic function. Other recent studies of CD161 function have indicated that this molecule may play a role in transendothelial migration. It has been shown that engagement of CD161 on NK-T cells appears to promote activation of ß1-integrins, resulting in enhanced migration of these T cells through endothelial monolayers [41 ]. DC are also highly motile cells, and it is possible that CD161 could be involved in the passage of maturing DC through lymphatic endothelial vessels during trafficking from sites of inflammation to lymph organs. The one specific consequence of stimulating DC via CD161, which has been demonstrated, is enhanced cytokine secretion. Cross-linking CD161 on human DC or monocytes in vitro has been shown to induce production of IL-1ß and IL-12 [19 ]. This would be consistent with CD161 having activating properties on at least some subsets of DC. It is also noteworthy because previous studies have shown that certain cognate interactions, i.e., interaction with microbial pathogens or cross-linking CD40 or MHC class II, can trigger DC to produce IL-12 [42 43 44 ]. As our data demonstrate that CD161 is expressed on mature DC, which are considered primed for interaction with T cells, we could speculate that perhaps CD161 functions as a novel accessory molecule involved in antigen presentation. By virtue of stimulating IL-12 production, ligation of CD161 on DC could potentially direct the development of a T helper cell type 1 (Th1) versus Th2 immune response. To address these issues, we are investigating whether signaling via CD161 on BM-derived DC leads to activation/differentiation of these DC directly and if CD161-mediated signaling synergizes with other know DC stimuli in modulating the antigen presenting functions of DC.

In summary, we have found that in the presence of FL, large numbers of DC can be generated, and these have a unique OX62+/CD161+ phenotype. It is not yet known whether these might represent a distinct subset of rat DC, i.e., myeloid versus lymphoid or plasmacytoid. Further, with the emerging understanding of differences in the role of DC1 versus DC2 subsets in stimulating T cells [45 ], it will be of interest to determine if these rat DC have unique functional capabilities. In turn, this could enhance our understanding of rat DC immunobiology and the development of translational studies of rat DC-based vaccines.


   ACKNOWLEDGEMENTS
 
This study was supported by a National of Institutes of Health grant (CA68550). We express appreciation to Jason Attanucci for additional technical support and to Hideho Okada for helpful discussions.

Received August 25, 2001; revised November 29, 2001; accepted November 30, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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