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Originally published online as doi:10.1189/jlb.0406257 on September 12, 2007

Published online before print September 12, 2007
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(Journal of Leukocyte Biology. 2007;82:1510-1518.)
© 2007 by Society for Leukocyte Biology

Disease-modifying capability of murine Flt3-ligand DCs in experimental autoimmune encephalomyelitis

Tracey L. Papenfuss*,1, Aaron P. Kithcart{dagger}, Nicole D. Powell{ddagger}, Melanie A. McClain{dagger}, Ingrid E. Gienapp{dagger}, Todd M. Shawler{dagger} and Caroline C. Whitacre{dagger}

* Departments of Veterinary Biosciences and
{dagger} Molecular Virology, Immunology and Medical Genetics, and
{ddagger} Section of Oral Biology, College of Dentistry, The Ohio State University, Columbus, Ohio, USA

1Correspondence: Department of Veterinary Biosciences, The Ohio State University, 370 Veterinary Medical Academic Building, 1900 Coffey Road, Columbus, OH 43210, USA. E-mail: papenfuss.1{at}osu.edu


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ABSTRACT
 
Dendritic cells (DCs) bridge the innate and adaptive immune response, are uniquely capable of priming naïve T cells, and play a critical role in the initiation and regulation of autoimmune and immune-mediated disease. At present, in vivo expansion of DC populations is accomplished primarily through the administration of the recombinant human growth factor fms-like tyrosine kinase 3 ligand (hFL), and in vitro DCs are generated using cytokine cocktails containing GM-CSF ± IL-4. Although hFL has traditionally been used in mice, differences in amino acid sequence and biological activity exist between murine FL (mFL) and hFL, and resultant DC populations differ in phenotype and immunoregulatory functional capabilities. This study developed and characterized mFL-generated DCs and determined the therapeutic capability of mFL DCs in the autoimmune disease experimental autoimmune encephalomyelitis (EAE). Our findings demonstrate that mFL and hFL expand splenic DCs equally in vivo but that mFL-expanded, splenic DCs more closely resemble normal, resting, splenic DCs. In addition, a novel method for generating mFL-derived bone marrow-derived DCs (BM-DCs) was developed, and comparison of mFL with hFL BM-DCs found mFL BM-DCs to be less mature (i.e., lower MHC Class II, CD80, and CD86) than hFL BM-DCs. These immature mFL DCs up-regulated costimulatory molecules in response to maturation stimuli LPS and TNF-{alpha}. Mature mFL BM-DCs were immunogenic and exacerbated the clinical disease course of EAE.

Key Words: dendritic cells • MS/EAE


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INTRODUCTION
 
Dendritic cells (DCs) are unique among APCs in their ability to prime naïve T lymphocytes. By bridging innate and adaptive immunity, DCs are positioned to influence the character of the immune response and impact inflammatory, autoimmune, and immune-mediated diseases. DC-based immunotherapy has been reported to be successful in transplantation, allergic, and autoimmune diseases [1 2 3 4 ]. However, the trace population of DCs in vivo and difficulty in isolating large numbers of DCs have previously limited investigations into DC biology. Recent advances have allowed the expansion and characterization of DC populations and facilitated the study of these cells. Most studies evaluate in vivo-expanded DCs or in vitro-differentiated bone marrow-derived DCs (BM-DCs), and methods used to generate these populations vary markedly. At present, in vivo expansion of DC populations (e.g., myeloid, lymphoid, and plasmacytoid DCs) is accomplished through the administration of the growth factor fms-like tyrosine kinase 3 ligand (FL), and in vitro DCs are traditionally generated with cytokine cocktails containing GM-CSF, with or without IL-4 [5 6 7 8 9 10 11 12 13 14 15 16 17 ]. The use of such different growth factors in the generation of in vivo versus in vitro DCs has limited specific comparison between these DC populations.

FL is a growth factor, which binds to receptor tyrosine kinase FL [fetal liver tyrosine kinase (flt), fetal liver kinase 2 (flk-2), or stem cell tyrosine kinase 1 (STK-1)] and is highly expressed on hematopoietic progenitor cells. Ligation of FL with its receptor results in expansion of DCs in vivo in mice, humans, and macaques with expanded DCs representing all DC lineages (i.e., myeloid, lymphoid, and plasmacytoid DCs) [6 , 10 , 13 , 18 19 20 21 22 23 24 25 26 ]. It is interesting that FL treatment has been shown to be capable of inducing regulatory and immunogenic DCs and may be a means to expand and study factors which influence whether a DC becomes immunogenic or regulatory [15 , 27 ]. Techniques for FL administration include daily s.c. injections of human FL (hFL) or murine FL (mFL), FL-secreting tumors, FL-expressing adenoviruses, or hydrodynamic-based gene delivery of FL DNA, although daily injection of hFL is by far the most common and well-characterized method of administration [6 , 11 12 13 14 15 16 17 ]. However, DCs generated in mice with a human (xenogeneic) growth factor may not model naturally occurring murine DC populations accurately. Indeed, hFL and mFL differ in sequence, form, and function, which likely influence resultant DC populations.

Although mFL and hFL share an overall amino acid sequence homology of 72%, there are differences in the cytoplasmic domains, biologically active isoforms, and overall potency between these two growth factors [15 , 18 , 28 29 30 ]. In addition, mFL-generated DCs have shown differences in cell surface marker expression (i.e., increased B220 expression) and cytokine production (i.e., decreased production of IL-6, IL-10, IFN-{gamma}, and TNF-{alpha}), which likely influence DC function and DC-T cell interactions [31 ]. At present, a direct comparison of hFL versus mFL DCs is lacking, likely as a result of limited availability and the large volumes required for injection of murine recombinant FL (mrFL). However, alternative methods of delivering mFL in vivo (e.g., mFL-secreting tumor lines and mFL cDNA plasmid delivery) are being used and necessitate a comparison of DCs expanded in vivo with mFL versus hFL.

Although the majority of studies have used FL to expand DCs in vivo, it has been reported recently that hFL and mFL can generate conventional (i.e., myeloid, CD11c+CD11b+CD8 {alpha}–; and lymphoid, CD11c+CD11b–CD8{alpha}+) and plasmacytoid (i.e., CD11c+CD11b–CD8{alpha}–) DCs from BM precursors (BM-PC), depending on culture conditions [10 , 32 , 33 ]. However, the generation of BM-DCs is by far accomplished predominantly with GM-CSF (±IL-4). Few studies explore BM-DCs generated with FL, and no studies to date compare the phenotype, function, and maturation of BM-DCs generated with different forms of FL [5 6 7 8 9 10 ]. The purpose of this study was to characterize more fully mFL-derived DCs phenotypically and functionally. As DC maturation status plays a critical role in determining whether GM-CSF DCs are immunogenic or more regulatory in nature, we explored the influence of the maturation status of mFL BM-DCs in a disease model of multiple sclerosis (MS) experimental autoimmune encephalomyelitis (EAE) [34 , 35 ].

We found that mFL is capable of expanding DCs in vivo and can be used to generate BM-DCs in vitro, similar to hFL. We also found that mrFL and mFL-derived BM-DCs were less mature than hFL BM-DCs and that all FL-derived BM-DCs were more similar to one another than to GM-CSF BM-DCs. Finally, immature mFL DCs up-regulated costimulatory molecules in response to maturation stimuli LPS/TNF-{alpha}, and such matured mFL BM-DCs were immunogenic and exacerbated the clinical disease course of EAE. Our findings indicate that mFL can be used in a novel approach to generate DCs in vitro, and the use of such murine growth factors may demonstrate subtle species-specific differences, which have important biological implications in mouse models of human disease.


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MATERIALS AND METHODS
 
Animals and treatments
Male and female 6- to 12-week-old C57BL/6 or B10.PL mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA) and used for in vivo DC expansion and as a source for BM-PC. In vivo DC expansion was carried out using recombinant hFL (kindly provided by Amgen, Thousand Oaks, CA, USA) and mFL-expressing melanoma cells (kindly provided by Dr. Paula Bryant, The Ohio State University, Columbus, OH, USA). hFL was administered once daily for 9 days (10 ng/mouse in 200 ul 1xPBS containing 0.1% mouse serum albumin) s.c. in the nape of the neck. Approximately 1–3 x 106 B16 melanoma cells (C57BL/6 background), transfected with mFL cDNA, using an MFG-retroviral vector, were injected s.c. and divided into two sites over the flanks. Mice were killed when tumor size measured 1–1.5 cm in diameter, as described previously [14 ].

Ex vivo DC isolation
Spleens (SPL) and lymph nodes (LN) were removed, and single cell suspensions were prepared. Cells were suspended in RPMI (supplemented with 25 mM HEPES, 2 mM L-glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin, and 10% FCS). Ex vivo CD11c+ DCs were purified by positive selection using CD11c+ magnetic bead separation (Miltenyi Biotec, Auburn, CA, USA). CD11c+ SPL and LN cells were evaluated for purity and phenotype by three-color flow cytometry using a FACSCalibur (BD Biosciences, San Jose, CA, USA).

BM-DC cultures
BM precursor cells (BM-PC) were flushed from femurs, tibias, and humeri of naïve C57BL/6 mice and cultured in the presence of supplemented RPMI with 20% mFL-containing supernatant, 200 ng/ml hFL, 100 ng/ml mrFL, or 20 ng/ml GM-CSF (Peprotech Inc., Rocky Hill, NJ, USA) [7 , 15 , 36 , 37 ]. Cells were plated at a concentration of 3–4 x 106 cells/ml in six-well tissue-culture plates, and 80% of the medium was replaced every 48–72 h. Cells were removed from culture at Days 7, 9, 11, 14, and 16, washed in cold PBS, and labeled using the antibodies described below. BM-DCs after 10–12 days of culture were used for phenotypic comparisons, DC maturation, and EAE studies.

Differentiation and maturation of BM-DCs
Days 10–12 BM-DCs generated in mFL-containing supernatant were used for DC maturation and EAE studies. To mature BM-DCs, immature BM-DCs (derived after 10–12 days of culture) were washed, replated at a concentration of 4–6 x 106 cells/ml, and cultured with maturation stimuli of TNF-{alpha} (5 µg/ml, Peprotech, Inc.) and LPS (1 µg/ml, Sigma-Aldrich, St. Louis, MO, USA). Cells were removed from culture after 24 h, washed in cold PBS, and evaluated phenotypically (flow cytometric labeling for CD40, CD80, CD86, and MHC Class II) and functionally in vivo by injection into naïve mice prior to EAE induction. Approximately 8–10 x 106 DCs were transferred i.v. to naïve recipient mice, 1 day prior to EAE induction.

Flow cytometry
In vivo- and in vitro-derived DCs were labeled and evaluated by three-color flow cytometry using combinations of the following antibodies conjugated directly: CD11b, CD11c, MHC Class II, CD8{alpha}, B220, CD80, CD86, programmed death ligand 1 (PD-L1), PD-L2, CD40, and inducible costimulatory molecule ligand (ICOSL) and the indirectly labeled antibody CD205 (all antibodies from BD Biosciences/PharMingen, San Diego, CA, USA). In vivo-expanded SPL DCs were evaluated for CD11c staining before and after CD11c+ purification. Cells were gated on CD11c+, or purified CD11c+ cells were labeled directly using the antibodies described above. In vitro-derived DCs were evaluated after various lengths in culture (Days 5, 7, 9, 11, 14, 16) for cell surface expression of phenotypic and costimulatory markers as described above. Appropriate isotype controls conjugated directly (BD Biosciences/PharMingen) were used for each three-color combination.

EAE induction
Mice were immunized s.c. in four sites over the back (left and right shoulder, left and right flank) with 100 µg myelin oligodendrocyte glycoprotein (MOG), 35–55 emulsified in equal volumes of CFA (containing 200 µg heat-killed Mycobacterium tuberculosis Jamaica strain). Mice received 200 ng pertussis toxin (PT) in 0.2 ml PBS (List Biological Laboratories, Campbell, CA, USA) i.p. at the time of immunization and 48 h later. Mice were evaluated daily for clinical signs of EAE for at least 30 days and scored as follows: 0, no clinical signs; +1, limp tail or waddling gait with tail tonicity; +2, ataxia or waddling gait with tail limpness; +3, partial hindlimb paralysis; +4, total hindlimbparalysis; and +5 moribund/death. Disease parameters of incidence, day of onset, peak disease score, and cumulative disease score [CDS; total and during acute disease (Days 0–21)] were calculated.


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RESULTS
 
Comparison of mFL and hFL in vivo-expanded DCs
hFL has been used to expand DCs in vivo in a variety of studies, but the use of mFL to expand DCs in vivo has been explored less thoroughly [6 , 9 10 11 12 13 , 16 , 20 21 22 23 24 , 38 , 39 ]. Initial studies took advantage of the fact that the use of mFL-secreting tumor cell lines circumvented the need for large quantities of mrFL required for in vivo studies. Mice receiving mFL or hFL showed an expanded number of CD11c+ cells within the SPL compared with vehicle-treated controls (Fig. 1 ). Similar findings were seen in the LN (data not shown). It is interesting that the phenotype of mFL and hFL splenic DC populations differed, and hFL-expanded DCs had decreased expression of CD11b, CD80, and CD86 compared with control mice and splenic mFL-derived CD11c+ DCs (Fig. 1B) . These data suggest that although hFL and mFL expand overall DC numbers similarly, the resultant DCs differ in phenotype and activation status. Specifically, mFL DCs resemble the phenotype of nonexpanded, normal (resting), splenic DCs more closely. To evaluate the use of this mFL-secreting cell line more thoroughly in the generation of in vitro DC populations, we developed an in vitro system with mFL-containing supernatant to generate BM-DCs.


Figure 1
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  		Figure 1. (A) In vivo expansion of splenic CD11c+ cells following mFL or hFL treatment or in vehicle controls (VEH) demonstrating relative percentage CD11c+ cells. Included are the percentage and the overall yield (in millions) of CD11c+ cells. (B) Phenotype of in vivo-expanded splenic CD11c+ cells following continuous mFL administration or daily hFL injection. Single cell suspensions of splenocytes were gated on CD11c+, and levels of CD11b, CD80, and CD86 were evaluated. Results are expressed in percent-positive cells ± SE; *, P < 0.05. hFL-expanded DCs showed decreased expression of CD11b, CD80, and CD86 compared with age-matched controls (CTRL). Data are from one of three (n=3) representative experiments.

Development of mFL BM-DCs and comparison with hFL and mrFL BM-DCs
Limited reports describe that FL (hFL and mrFL) can be used to generate BM-DCs, which have different functions and potentially more regulatory than GM-CSF-derived BM-DCs [10 , 19 , 25 , 26 , 32 ]. Adapting the mFL used for in vivo expansion to an in vitro system, we observed that over 95% CD11c+ cells were generated from BM-PC after 14 days of culture (Fig. 2A ) with a dramatic shift in CD11c expression as early as day 11 of culture (Fig. 2B) . CD11c expression in mrFL- and mFL-derived DCs was higher than in hFL-derived DCs. We then compared BM-DCs generated with mFL-containing supernatant (mFL) to BM-DCs generated with mrFL or hFL. Overall expression of CD11b, B220, and CD8{alpha} was similar among all groups, and no significant differences were between mrFL- and mFL-stimulated cell populations.


Figure 2
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  		Figure 2. (A) Kinetics of CD11c+ expression in BM-PC following in vitro exposure to mFL-containing supernatant for 16 days. (B) Overlay of CD11c+ expression of 12-day BM-DCs generated in the presence of hFL, mrFL, or mFL. All BM-PC were cultured with 200 ng/ml hFL-, 100 ng/ml mrFL-, or 20% mFL-containing supernatant, and Day 12 cell populations were evaluated by flow cytometry. (C) Phenotypic expression of CD11b, B220, and CD8{alpha} on hFL-, mrFL-, and mFL-derived CD11c+ BM-DCs. Data are representative of five individual experiments. APC, Allophycocyanin.

Functional evaluation and comparison of hFL, mrFL, and mFL BM-DCs cocultured with T cells
To understand whether BM-DCs generated with hFL, mrFL, and mFL differed functionally, we evaluated their ability to stimulate proliferation of naïve T cells and the cytokine production of BM-DCs cultured alone or in the presence of T cells. Naïve 2D2 MOG-TCR-specific CD4+ T cells were cultured with DC (at a ratio of 5:1, T cells:DC). All FL groups stimulated similar proliferation of T cells, regardless of whether DCs (i.e., LPS) or T cells (i.e., anti-CD3) were stimulated (Fig. 3A and 3B ). Peak proliferation occurred at 72 h of culture with both in vitro stimuli. MOG stimulation demonstrated an identical pattern of proliferation as anti-CD3 stimulation (data not shown), and low levels of proliferation were observed in all of the unstimulated groups. Levels of TNF-{alpha} and IFN-{gamma} were evaluated in DC:T cell co-cultures following stimulation of DCs (i.e., LPS) or T cells (i.e., anti-CD3; Fig. 3C and 3D ). The increase in TNF-{alpha} was highest for mFL, regardless of whether LPS or anti-CD3 was used for stimulation, perhaps reflecting the effect of the tumor. IFN-{gamma} production was increased only in the anti-CD3-stimulated group, indicating T cells as the primary source for this cytokine. T cells co-cultured with mrFL-derived BM-DCs demonstrated the greatest increase in IFN-{gamma} compared with hFL and mFL BM-DCs. These studies suggest that hFL, mrFL, and mFL BM-DCs were functionally comparable in their ability to influence naïve T cell proliferation and demonstrated only subtle differences in production of the cytokines TNF-{alpha} and IFN-{gamma}. The differences in relative cytokine production suggested that these differentially derived BM-DCs may differ in their relative activation status and their ability to influence T cell cytokine production.


Figure 3
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  		Figure 3. Proliferative responses and cytokine production in BM-DC:T cell cocultures. BM-DCs were cocultured with naïve 2D2 MOG-TCR CD4+ T cells (ratio 1:5), cultured for 72 h in the presence of LPS or anti-CD3. Plates evaluated for proliferation were pulsed with 3H-thymidine in the last 18 h of culture (A, LPS; B, anti-CD3). The levels of (C) TNF-{alpha} and (D) IFN-{gamma} in 72 h culture supernatants were determined by cytometric bead array and reported as fold-increase over media levels for LPS and anti-CD3. Data are representative of three individual experiments.

Phenotypic comparison of activation markers on BM-DC populations
To determine whether these FL BM-DC populations differed in their baseline level of activation, we evaluated MHC Class II and costimulatory (stimulatory and inhibitory) marker expression (CD80, CD86, PD-L1, and PD-L2) on hFL-, mrFL-, mFL-, and GM-CSF-derived BM-DCs. Levels of MHC Class II and all costimulatory markers were generally comparable among all FL groups (Fig. 4 ). GM-CSF BM-DCs had significantly different expression patterns, with decreased expression of MHC Class II and PD-L2 and increased levels of CD80, CD86, and PD-L1 compared with all FL groups (Fig. 4) . Similar to in vivo differences in hFL and mFL DC phenotype, greater numbers of hFL BM-DCs express higher MHC Class II, CD80, and CD86 compared with mrFL and mFL BM-DCs, suggesting that hFL DCs are more mature than mrFL or mFL BM-DCs. Expression patterns were similar between mrFL and mFL BM-DCs, with the exception of PD-L2. mFL BM-DCs have increased numbers of cells expressing PD-L2, a costimulatory molecule with inhibitory and potentially immunoregulatory function. As mFL BM-DCs demonstrated an immature phenotype in vivo and in vitro and expressed increased PD-L2 expression, we next evaluated whether these DCs were functional and capable of maturation.


Figure 4
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  		Figure 4. Phenotypic and costimulatory marker expression of MHC Class II, CD80, CD86, PD-L1, and PD-L2 in BM-DC populations after 12 days in culture. BM-PC were cultured with media supplemented with 200 ng/ml hFL-, 100 ng/ml mrFL-, and 20% mFL-containing supernatant and 20 ng/ml GM-CSF (with 70% media changes every 2–3 days) and evaluated by flow cytometry at Day 12 of culture. Data shown are representative of five individual experiments.

Function and maturation of mFL BM-DCs
mFL BM-DCs were exposed to maturation stimuli TNF-{alpha} ± LPS for 24 h. TNF-{alpha} exposure was sufficient to induce increased expression of all costimulatory molecules (i.e., CD80, CD86, CD40, PD-L1, and ICOSL; Fig. 5 ). Exposure to a combination of LPS and TNF-{alpha} resulted in additional increases in maturation markers CD80, CD86, and CD40 but a decrease in ICOSL expression. Although PD-L2 expression was increased over media-exposed BM-DCs, the overall expression of PD-L2 on cells was low (<10%, data not shown). These results demonstrate that BM-DCs generated with mFL were immature and capable of maturation when cultured with maturation stimuli.


Figure 5
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  		Figure 5. BM-DCs cultured in the presence of different maturation stimuli demonstrate distinct phenotypic differences in costimulatory molecule expression. Immature (10–12 days) BM-DCs were cultured in the presence of 10 µg/ml MOG 35–55 and cultured in the presence of media (immature) or with maturation factors LPS ± TNF-{alpha} (1 µg/ml each) for 18–24 h. Cells were washed and evaluated immediately after culture (gated on CD11c) for the expression of costimulatory molecules CD80, CD86, CD40, PD-L1, PD-L2, and ICOSL. Bold lines represent LPS + TNF-{alpha}; thin lines, TNF-{alpha} alone; shaded areas, media-treated cells, respectively. Figures are representative of three independent experiments.

Differentially matured DCs influence EAE
To determine whether mFL BM-DCs could influence the nature of an immune response in vivo, immature and mature mFL BM-DCs (treated with TNF-{alpha} and LPS) were administered to naïve mice prior to the induction of EAE. DCs (8–10x106) were transferred i.v. to naïve C57BL/6 mice 1 day before immunization with MOG 35–55 plus adjuvants. Mice receiving immature BM-DCs had less severe disease, and those receiving mature BM-DCs had increased disease compared with control mice receiving no DCs (i.e., EAE alone; Fig. 6 ). Mice receiving matured BM-DCs prior to immunization had significantly increased peak disease scores (3.5±1.3) and CDS [overall (41.1±15.5) and in the acute phase of disease (26.8±10.1)] compared with immature mFL BM-DCs [peak disease score (1.9±0.7) and CDS overall (20.2±7.7) and acute (13.0±4.9), respectively]. There was no difference in incidence or day of onset among any of the groups (Fig. 6) . Although transfer of immature BM-DCs appeared to protect against EAE (compared with EAE-only mice receiving no DCs), this did not achieve statistical significance. It is interesting that exposure of DCs to MOG peptide alone appears to mature DCs partially, resulting in EAE severity similar to mice receiving no DCs, and TNF-{alpha} or LPS alone did not produce the marked increase in severity in acute EAE as seen in LPS + TNF-{alpha}-matured DCs (data not shown). Therefore, the maturation status of mFL BM-DCs influenced the clinical outcome of EAE. Taken together, these findings indicate that mFL generates DCs, which are immature (in vivo and in vitro), and that immature BM-DCs, upon maturation, become immunogenic and modulate the disease course of the autoimmune disease EAE.


Figure 6
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  		Figure 6. Differential effects of mature and immature Day 10 mFL BM-DC on EAE. BM-DCs were pulsed in the presence of MOG (10 µg/ml), with or without the maturation agents LPS and TNF-{alpha} (both at 1 µg/ml) for 18–24 h, and 8–10 million DCs were transferred i.v. to naïve recipients. One day later, recipients were immunized with MOG 35–55 and adjuvant and monitored for the development of EAE, as described in Materials and Methods. Clinical parameters of incidence, day of onset, peak score, and CDS (acute, Days 0–21, and overall) are shown; *, statistical significance (P<0.05) comparing mice that received mature versus immature DCs. Data are representative of three individual experiments with an n = 5 per group.


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DISCUSSION
 
The majority of work investigating DC maturation status has used GM-CSF BM-DCs, and few studies investigate the maturation status and functional nature of FL DCs. However, the method of DC generation plays an important role in the phenotype and function of DCs. Functional activities of DCs are dependent, not only on their lineage or tissue source but also on their state of differentiation, maturation, and activation [40 , 41 ]. In essence, terminally differentiated, mature DCs can induce T cell responses efficiently, and immature DCs are involved in the maintenance of peripheral tolerance [34 ]. The purpose of this study was to evaluate the use of mFL to generate DCs, compare BM-DCs generated with different sources of FL, and determine whether the maturation status of mFL DCs would influence a disease model, EAE.

hFL injection has been the primary method used to expand DCs in vivo [6 , 13 , 18 19 20 , 22 ]. Yet, recent data indicate that differences exist in the properties of hFL and mFL and the biological activity of resultant DCs [15 , 18 , 28 29 30 31 ]. Our initial studies demonstrated that hFL and mFL were equally capable of expanding DCs in vivo in the SPL compartment but that hFL- and mFL-derived DCs differed in phenotype (Fig. 1) . DCs expanded with mFL were indistinguishable from control, unexpanded DCs for CD11b, CD80, and CD86 expression. In contrast, splenic DCs expanded with hFL showed reduced levels of these markers, most notably CD86. Thus, mFL may more accurately produce normal mouse physiologic and immunologic responses and may generate more immature DCs. From these results, we explored the influence of mFL on the generation of BM-DCs. It is important that the use of a murine growth factor may be a more physiologically relevant alternative to xenogeneic hFL when evaluating subtle differences in DC populations within mouse models.

Currently, direct comparisons of hFL versus mFL DCs in vivo are likely limited as a result of the large quantities required for injections and expense involved with administration of mrFL. Alternative methods for delivery of mFL (e.g., FL-secreting tumors, FL-expressing adenoviruses, or hydrodynamic-based gene delivery of FL DNA) preclude direct comparison with injection of recombinant hFL [6 , 11 12 13 14 15 16 17 ]. However, as investigators begin to use mFL-secreting tumor lines and mFL plasmids, an understanding of the phenotypic and functional differences between hFL and mFL becomes critical in the evaluation of DC populations and use of DC-based immunotherapy [17 , 42 43 44 45 46 ].

To develop a biologically similar in vitro correlate to the in vivo mFL delivery system, BM-PC were cultured in the presence of mFL-containing (tumor-derived) supernatant or recombinant hFL. Although it can be argued that use of a tumor-based mFL delivery system may influence DC phenotype or function, it has been shown that mFL-secreting B16 melanoma cells elicit comparable effects on hematopoietic populations compared with injections of mrFL, that resultant DCs function efficiently as stimulators of MLRs, and that these DCs are capable of migration and T cell priming in vivo [6 , 13 , 14 , 47 ]. To address the issue that a tumor-derived supernatant may contain additional factors, which influence BM-DC phenotype, we compared (phenotypically and functionally) mFL and hFL BM-DC populations with those cultured in the presence of mrFL.

The expression patterns of several phenotypic (e.g., CD11c, CD11b, MHC Class II) and costimulatory markers (CD80, CD86, and PD-L1) on DCs derived from mFL and mrFL exposure suggest that the use of a mFL-containing supernatant generates BM-DCs, which are more similar to mrFL than to hFL (Figs. 2 and 4) . In contrast to what was observed in vivo in the SPL with hFL administration, hFL BM-DCs had increased numbers of cells expressing CD80 and CD86 (Fig. 4) , supporting that BM-DCs exposed to hFL in vitro are more mature than DCs exposed to mFL or mrFL. Subtle differences in B220 and CD8{alpha} expression were seen among these three populations (Fig. 2) , suggesting that hFL, mFL, and mrFL may differ in their ability to generate plasmacytoid (or other) DC populations, and work is currently underway to investigate these differences [15 , 48 ].

As expected, IFN-{gamma} production occurs in DC:T cell cocultures only upon stimulation of T cells with anti-CD3 (Fig. 3D) , and our results suggest that subtle differences exist in IFN-{gamma} production, depending on the method used to generate BM-DCs. In contrast to IFN-{gamma}, production of TNF-{alpha} is similar, regardless of whether DC or T cell stimuli are used (Fig. 3C) . These results suggest that co-culturing BM-DC populations with T cells is sufficient to generate maximal TNF-{alpha} production, as further maturation of BM-DCs with LPS did not increase TNF-{alpha} production further (Fig. 3B) . The source of TNF-{alpha} (i.e., T cells or DCs) and importance of TNF-{alpha} production in our system are not known, but TNF-{alpha} has been shown to have a decidedly pleiotropic effect in the pathogensis of autoimmune disease. TNF-{alpha} has been associated with the promotion of proinflammatory Th1 responses/CNS pathology and protection from relapses [49 , 50 ]. Recent studies have also demonstrated that TNF-{alpha} can generate "semi-mature" DCs, which have protective effects in EAE [51 ]. Therefore, further studies are necessary to determine the influence of cytokines such as TNF-{alpha} and IFN-{gamma} on the DC and T cell compartments in the pathogenesis of disease.

Expression of MHC Class II and costimulatory molecules CD80 and CD86 are used to identify the maturation status of DC populations phenotypically, and results from Figure 4 suggest that mrFL and mFL are less mature (based on CD80 and CD86 expression) than hFL DCs. GM-CSF DCs differed dramatically from FL-derived BM-DCs in phenotype. GM-CSF DCs had a broader population of cells expressing MHC Class II, CD80, CD86, and PD-L1 and greater overall intensity of expression of CD80, CD86, and PD-L1. Expression of the inhibitory costimulatory molecule PD-L2 was absent. Thus, more of the GM-CSF BM-DCs are mature than any of the FL BM-DCs, and they express greater stimulatory and inhibitory costimulatory molecules.

Evaluation of the inhibitory costimulatory molecule PD-L2 in the FL-derived BM-DC populations demonstrated the most significant differences. The number of cells expressing PD-L2 was increased in mFL BM-DCs, relative to the other FL-derived DC populations. PD-L2 is an inhibitory costimulatory molecule, and it has been shown that blockade of PD-L2 or its ligand PD-1 will exacerbate EAE through increased T cell activation, IFN-{gamma} production, and increased leukocyte infiltration into the CNS [52 ]. The precise role of PD-L2 expression on the mFL BM-DCs is not known but may have subtle influences on the interaction of BM-DCs with T cells. However, PD-L2 does not appear to alter the ability of mFL BM-DCs to cause T cell proliferation when compared with hFL or mrFL (Fig. 3A and 3B) , although Figure 3D suggests a slight decrease in IFN-{gamma} production of T cells in mFL BM-DCs compared with mrFL-derived DCs.

Taken together, our findings support that DCs expanded with mFL and mrFL are similar phenotypically and appear less mature than hFL DCs. Functionally, hFL-, mrFL-, and mFL-derived BM-DCs do not appear to differ in their ability to cause proliferation of naïve, antigen-specific T cell populations, but they may have subtle differences in cytokine generation. We found that mFL BM-DCs were capable of maturation and that TNF-{alpha} up-regulated CD80, CD86, CD40, PD-L1, and PD-L2, and the addition of LPS further increased the levels of activation markers CD80, CD86, and CD40. One interesting observation is that the addition of LPS actually decreased ICOSL expression compared with TNF-{alpha} alone. High expression of ICOSL has been demonstrated to promote the development of Th2 cells in particular, and its expression on plasmacytoid DCs was shown to play a role in the generation of regulatory T cells [53 , 54 ]. Thus, the use of LPS for maturation may not only up-regulate immunogenic signals such as CD80 and CD86 but may also decrease immunoregulatory molecules such as ICOSL.

To evaluate whether mFL BM-DCs behaved similarly to other reported immature DCs, the protective effects of immature and matured mFL BM-DCs were evaluated in vivo in EAE. Mice receiving mature mFL BM-DCs prior to immunization had significantly increased CDS and peak disease score compared with recipients of immature mFL BM-DCs (Fig. 6) . Transfer of immature BM-DCs was suggestive of protection from EAE but did not achieve statistical significance. The lack of definitive protection from EAE may be a result of the presence of strong, activating stimuli required for EAE immunization. CFA and PT are potent activating stimuli, which drive a Th1 response, resulting in EAE clinical disease. An immature DC in the face of activating stimuli may have transitioned to a more immunogenic DC phenotype in vivo following immunization.

In summary, our results demonstrate that mFL and hFL DCs differ and that mFL is a viable means to generate BM-DCs. Immature mFL BM-DCs modulate T cell proliferation and cytokine production and are capable of maturation. It is important that the maturation status of mFL BM-DCs influence the clinical disease course of autoimmune disease. Our findings indicate that mFL is a novel method to generate DCs in vitro, and the use of such murine growth factors may demonstrate subtle species-specific differences, which influence mouse models of human disease.


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ACKNOWLEDGEMENTS
 
Support for this work was supplied by NIH ROI NS048316-03 and the National Multiple Sclerosis Society (NMSS) RG 3272-A-5 and F61555-A-1.

Received April 7, 2006; revised August 23, 2007; accepted August 26, 2007.


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REFERENCES
 
    1
  1. Raimondi, G., Thomson, A. W. (2005) Dendritic cells, tolerance and therapy of organ allograft rejection Contrib. Nephrol. 146,105-120[Medline]
    2. 2
  2. O’Neill, D. W., Adams, S., Bhardwaj, N. (2004) Manipulating dendritic cell biology for the active immunotherapy of cancer Blood 104,2235-2246[Abstract/Free Full Text]
    3. 3
  3. Adorini, L. (2003) Tolerogenic dendritic cells induced by vitamin D receptor ligands enhance regulatory T cells inhibiting autoimmune diabetes Ann. N. Y. Acad. Sci. 987,258-261[Medline]
    4. 4
  4. Figdor, C. G., de Vries, I. J., Lesterhuis, W. J., Melief, C. J. (2004) Dendritic cell immunotherapy: mapping the way Nat. Med. 10,475-480[CrossRef][Medline]
    5. 5
  5. Banchereau, J., Briere, F., Caux, C., Davoust, J., Lebecque, S., Liu, Y. J., Pulendran, B., Palucka, K. (2000) Immunobiology of dendritic cells Annu. Rev. Immunol. 18,767-811[CrossRef][Medline]
    6. 6
  6. 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]
    7. 7
  7. Inaba, K., Inaba, M., Romani, N., Aya, H., Deguchi, M., Ikehara, S., Muramatsu, S., Steinman, R. M. (1992) Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor J. Exp. Med. 176,1693-1702[Abstract/Free Full Text]
    8. 8
  8. Lutz, M. B., Kukutsch, N., Ogilvie, A. L., Rossner, S., Koch, F., Romani, N., Schuler, G. (1999) An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow J. Immunol. Methods 223,77-92[CrossRef][Medline]
    9. 9
  9. Huang, Y. M., Hussien, Y., Yarilin, D., Xiao, B. G., Liu, Y. J., Link, H. (2001) Interferon-β induces the development of type 2 dendritic cells Cytokine 13,264-271[CrossRef][Medline]
    10. 10
  10. Gilliet, M., Boonstra, A., Paturel, C., Antonenko, S., Xu, X. L., Trinchieri, G., O’Garra, A., Liu, Y. J. (2002) The development of murine plasmacytoid dendritic cell precursors is differentially regulated by FLT3-ligand and granulocyte/macrophage colony-stimulating factor J. Exp. Med. 195,953-958[Abstract/Free Full Text]
    11. 11
  11. Brasel, K., McKenna, H. J., Charrier, K., Morrissey, P. J., Williams, D. E., Lyman, S. D. (1997) Flt3 ligand synergizes with granulocyte-macrophage colony-stimulating factor or granulocyte colony-stimulating factor to mobilize hematopoietic progenitor cells into the peripheral blood of mice Blood 90,3781-3788[Abstract/Free Full Text]
    12. 12
  12. Brasel, K., McKenna, H. J., Morrissey, P. J., Charrier, K., Morris, A. E., Lee, C. C., Williams, D. E., Lyman, S. D. (1996) Hematologic effects of flt3 ligand in vivo in mice Blood 88,2004-2012[Abstract/Free Full Text]
    13. 13
  13. Maraskovsky, E., Pulendran, B., Brasel, K., Teepe, M., Roux, E. R., Shortman, K., Lyman, S. D., McKenna, H. J. (1997) Dramatic numerical increase of functionally mature dendritic cells in FLT3 ligand-treated mice Adv. Exp. Med. Biol. 417,33-40[Medline]
    14. 14
  14. Mach, N., Gillessen, S., Wilson, S. B., Sheehan, C., Mihm, M., Dranoff, G. (2000) Differences in dendritic cells stimulated in vivo by tumors engineered to secrete granulocyte-macrophage colony-stimulating factor or Flt3-ligand Cancer Res. 60,3239-3246[Abstract/Free Full Text]
    15. 15
  15. 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]
    16. 16
  16. Shaw, S. G., Maung, A. 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]
    17. 17
  17. He, Y., Pimenov, A. A., Nayak, J. V., Plowey, J., Falo, L. D., Jr, Huang, L. (2000) Intravenous injection of naked DNA encoding secreted flt3 ligand dramatically increases the number of dendritic cells and natural killer cells in vivo Hum. Gene Ther. 11,547-554[CrossRef][Medline]
    18. 18
  18. Shurin, M. R., Esche, C., Lotze, M. T. (1998) FLT3: receptor and ligand. Biology and potential clinical application Cytokine Growth Factor Rev. 9,37-48[CrossRef][Medline]
    19. 19
  19. Karsunky, H., Merad, M., Cozzio, A., Weissman, I. L., Manz, M. G. (2003) Flt3 ligand regulates dendritic cell development from Flt3+ lymphoid and myeloid-committed progenitors to Flt3+ dendritic cells in vivo J. Exp. Med. 198,305-313[Abstract/Free Full Text]
    20. 20
  20. Maraskovsky, E., Daro, E., Roux, E., Teepe, M., Maliszewski, C. R., Hoek, J., Caron, D., Lebsack, M. E., McKenna, H. J. (2000) In vivo generation of human dendritic cell subsets by Flt3 ligand Blood 96,878-884[Abstract/Free Full Text]
    21. 21
  21. Pulendran, B., Lingappa, J., Kennedy, M. K., Smith, J., Teepe, M., Rudensky, A., Maliszewski, C. R., Maraskovsky, E. (1997) Developmental pathways of dendritic cells in vivo: distinct function, phenotype, and localization of dendritic cell subsets in FLT3 ligand-treated mice J. Immunol. 159,2222-2231[Abstract/Free Full Text]
    22. 22
  22. 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]
    23. 23
  23. Pulendran, B., Banchereau, J., Burkeholder, S., Kraus, E., Guinet, E., Chalouni, C., Caron, D., Maliszewski, C., Davoust, J., Fay, J., Palucka, K. (2000) Flt3-ligand and granulocyte colony-stimulating factor mobilize distinct human dendritic cell subsets in vivo J. Immunol. 165,566-572[Abstract/Free Full Text]
    24. 24
  24. Teleshova, N., Jones, J., Kenney, J., Purcell, J., Bohm, R., Gettie, A., Pope, M. (2004) Short-term Flt3L treatment effectively mobilizes functional macaque dendritic cells J. Leukoc. Biol. 75,1102-1110[Abstract/Free Full Text]
    25. 25
  25. D’Amico, A., Wu, L. (2003) The early progenitors of mouse dendritic cells and plasmacytoid predendritic cells are within the bone marrow hemopoietic precursors expressing Flt3 J. Exp. Med. 198,293-303[Abstract/Free Full Text]
    26. 26
  26. Morse, M. A., Nair, S., Fernandez-Casal, M., Deng, Y., St Peter, M., Williams, R., Hobeika, A., Mosca, P., Clay, T., Cumming, R. I., Fisher, E., Clavien, P., Proia, A. D., Niedzwiecki, D., Caron, D., Lyerly, H. K. (2000) Preoperative mobilization of circulating dendritic cells by Flt3 ligand administration to patients with metastatic colon cancer J. Clin. Oncol. 18,3883-3893[Abstract/Free Full Text]
    27. 27
  27. McQualter, J. L., Darwiche, R., Ewing, C., Onuki, M., Kay, T. W., Hamilton, J. A., Reid, H. H., Bernard, C. C. (2001) Granulocyte macrophage colony-stimulating factor: a new putative therapeutic target in multiple sclerosis J. Exp. Med. 194,873-882[Abstract/Free Full Text]
    28. 28
  28. Broxmeyer, H. E., Lu, L., Cooper, S., Ruggieri, L., Li, Z. H., Lyman, S. D. (1995) Flt3 ligand stimulates/costimulates the growth of myeloid stem/progenitor cells Exp. Hematol. 23,1121-1129[Medline]
    29. 29
  29. Lyman, S. D., James, L., Johnson, L., Brasel, K., de Vries, P., Escobar, S. S., Downey, H., Splett, R. R., Beckmann, M. P., McKenna, H. J. (1994) Cloning of the human homologue of the murine flt3 ligand: a growth factor for early hematopoietic progenitor cells Blood 83,2795-2801[Abstract/Free Full Text]
    30. 30
  30. O’Keeffe, M., Hochrein, H., Vremec, D., Pooley, J., Evans, R., Woulfe, S., Shortman, K. (2002) Effects of administration of progenipoietin 1, Flt-3 ligand, granulocyte colony-stimulating factor, and pegylated granulocyte-macrophage colony-stimulating factor on dendritic cell subsets in mice Blood 99,2122-2130[Abstract/Free Full Text]
    31. 31
  31. 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]
    32. 32
  32. Liu, Y. J. (2001) Dendritic cell subsets and lineages, and their functions in innate and adaptive immunity Cell 106,259-262[CrossRef][Medline]
    33. 33
  33. Naik, S. H., Proietto, A. I., Wilson, N. S., Dakic, A., Schnorrer, P., Fuchsberger, M., Lahoud, M. H., O’Keeffe, M., Shao, Q. X., Chen, W. F., Villadangos, J. A., Shortman, K., Wui, L. (2005) Cutting edge: generation of splenic CD8+ and CD8– dendritic cell equivalents in Fms-like tyrosine kinase 3 ligand bone marrow cultures J. Immunol. 174,6592-6597[Abstract/Free Full Text]
    34. 34
  34. Tan, J. K., O’Neill, H. C. (2005) Maturation requirements for dendritic cells in T cell stimulation leading to tolerance versus immunity J. Leukoc. Biol. 78,319-324[Abstract/Free Full Text]
    35. 35
  35. Woltman, A. M., van Kooten, C. (2003) Functional modulation of dendritic cells to suppress adaptive immune responses J. Leukoc. Biol. 73,428-441[Abstract/Free Full Text]
    36. 36
  36. Lutz, M. B., Kukutsch, N. A., Menges, M., Rossner, S., Schuler, G. (2000) Culture of bone marrow cells in GM-CSF plus high doses of lipopolysaccharide generates exclusively immature dendritic cells which induce alloantigen-specific CD4 T cell anergy in vitro Eur. J. Immunol. 30,1048-1052[CrossRef][Medline]
    37. 37
  37. Brasel, K., De Smedt, T., Smith, J. L., Maliszewski, C. R. (2000) Generation of murine dendritic cells from flt3-ligand-supplemented bone marrow cultures Blood 96,3029-3039[Abstract/Free Full Text]
    38. 38
  38. Kohm, A.P., Carpentier, P.A., Miller, S.D. (2003) Regulation of experimental autoimmune encephalomyelitis (EAE) by CD4+CD25+ regulatory T cells Novartis Found. Symp. 252,45-52[Medline]
    39. 39
  39. Baulieu, E., Schumacher, M. (2000) Progesterone as a neuroactive neurosteroid, with special reference to the effect of progesterone on myelination Steroids 65,605-612[CrossRef][Medline]
    40. 40
  40. O’Neill, H. C., Wilson, H. L., Quah, B., Abbey, J. L., Despars, G., Ni, K. (2004) Dendritic cell development in long-term spleen stromal cultures Stem Cells 22,475-486[Abstract/Free Full Text]
    41. 41
  41. Mahnke, K., Schmitt, E., Bonifaz, L., Enk, A. H., Jonuleit, H. (2002) Immature, but not inactive: the tolerogenic function of immature dendritic cells Immunol. Cell Biol. 80,477-483[CrossRef][Medline]
    42. 42
  42. Lugo-Villarino, G., Ito, S., Klinman, D. M., Glimcher, L. H. (2005) The adjuvant activity of CpG DNA requires T-bet expression in dendritic cells Proc. Natl. Acad. Sci. USA 102,13248-13253[Abstract/Free Full Text]
    43. 43
  43. Shinohara, M. L., Lu, L., Bu, J., Werneck, M. B., Kobayashi, K. S., Glimcher, L. H., Cantor, H. (2006) Osteopontin expression is essential for interferon-{alpha} production by plasmacytoid dendritic cells Nat. Immunol. 7,498-506[CrossRef][Medline]
    44. 44
  44. Towne, C. F., York, I. A., Watkin, L. B., Lazo, J. S., Rock, K. L. (2007) Analysis of the role of bleomycin hydrolase in antigen presentation and the generation of CD8 T cell responses J. Immunol. 178,6923-6930[Abstract/Free Full Text]
    45. 45
  45. Song, Y. K., Liu, F., Zhang, G., Liu, D. (2002) Hydrodynamics-based transfection: simple and efficient method for introducing and expressing transgenes in animals by intravenous injection of DNA Methods Enzymol. 346,92-105[Medline]
    46. 46
  46. Liu, F., Song, Y., Liu, D. (1999) Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA Gene Ther. 6,1258-1266[CrossRef][Medline]
    47. 47
  47. Mempel, T. R., Henrickson, S. E., Von Andrian, U. H. (2004) T-cell priming by dendritic cells in lymph nodes occurs in three distinct phases Nature 427,154-159[CrossRef][Medline]
    48. 48
  48. Morelli, A. E., Thomson, A. W. (2003) Dendritic cells: regulators of alloimmunity and opportunities for tolerance induction Immunol. Rev. 196,125-146[CrossRef][Medline]
    49. 49
  49. Kollias, G., Kontoyiannis, D. (2002) Role of TNF/TNFR in autoimmunity: specific TNF receptor blockade may be advantageous to anti-TNF treatments Cytokine Growth Factor Rev. 13,315-321[CrossRef][Medline]
    50. 50
  50. Xiao, B. G., Link, H. (1999) Antigen-specific T cells in autoimmune diseases with a focus on multiple sclerosis and experimental allergic encephalomyelitis Cell. Mol. Life Sci. 56,5-21[CrossRef][Medline]
    51. 51
  51. Menges, M., Rossner, S., Voigtlander, C., Schindler, H., Kukutsch, N. A., Bogdan, C., Erb, K., Schuler, G., Lutz, M. B. (2002) Repetitive injections of dendritic cells matured with tumor necrosis factor {alpha} induce antigen-specific protection of mice from autoimmunity J. Exp. Med. 195,15-21[CrossRef][Medline]
    52. 52
  52. Salama, A. D., Chitnis, T., Imitola, J., Ansari, M. J., Akiba, H., Tushima, F., Azuma, M., Yagita, H., Sayegh, M. H., Khoury, S. J. (2003) Critical role of the programmed death-1 (PD-1) pathway in regulation of experimental autoimmune encephalomyelitis J. Exp. Med. 198,71-78[Abstract/Free Full Text]
    53. 53
  53. Ito, T., Yang, M., Wang, Y. H., Lande, R., Gregorio, J., Perng, O. A., Qin, X. F., Liu, Y. J., Gilliet, M. (2007) Plasmacytoid dendritic cells prime IL-10-producing T regulatory cells by inducible costimulator ligand J. Exp. Med. 204,105-115[Abstract/Free Full Text]
    54. 54
  54. McAdam, A. J., Chang, T. T., Lumelsky, A. E., Greenfield, E. A., Boussiotis, V. A., Duke-Cohan, J. S., Chernova, T., Malenkovich, N., Jabs, C., Kuchroo, V. K., Ling, V., Collins, M., Sharpe, A. H., Freeman, G. J. (2000) Mouse inducible costimulatory molecule (ICOS) expression is enhanced by CD28 costimulation and regulates differentiation of CD4+ T cells J. Immunol. 165,5035-5040[Abstract/Free Full Text]




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