Proteins of the Ikaros family control dendritic cell maturation required to induce optimal Th1 T cell differentiation

Mojgan Movassagh, Diego Laderach and Anne Galy1

Barbara Ann Karmanos Cancer Institute and Department of Immunology and Microbiology, Wayne State University, Detroit, MI 48201 USA 1 INSERM U362, Institut Gustave Roussy, Villejuif and Généthon, Evry, France

Correspondence to: A. Galy; E-mail: galy{at}genethon.fr
Transmitting editor: G. Trinchieri


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Ikaros proteins are pleiotropic regulators of hematopoiesis and are critically required for the production of lymphocyte and dendritic cell (DC) lineages in mice. Here, we asked if Ikaros proteins could also play a role in the late stages of dendritic cell differentiation. Nuclear Ikaros proteins were up-regulated during the in vitro differentiation of human monocytes into mature DC, suggesting potential implications in this process. To address this question, a dominant negative mutant Ikaros isoform IK7 was over-expressed by retroviral gene transfer in human DC precursor cells, to interfere with the function of Ikaros family members during DC development. Expression of IK7 in CD34+ cells inhibited the production of IL-12-producing APCs. The resulting progeny of CD34+ cells and in particular, committed CD1a+ DC or CD14+ cell-derived DC, expressed low levels of MHC class II antigens and of the CD83 maturation marker on the cell surface. Such IK7-expressing DC induced naïve allogeneic T cells to produce Th2 cytokines. Our results therefore delineate a new role for Ikaros family members, showing that normal levels of Ikaros proteins are essential in DC to regulate the terminal stages of maturation and the capacity to induce optimal Th1 T cell responses.

Keywords: antigen presentation, dendritic cells, human, transcription factors


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Dendritic cells (DC) constitute a complex system of antigen presenting cells (APC) with the paradoxical role of maintaining tolerance to self while inducing immune responses to foreign antigens (1). Animal models or human cell systems have helped to understand the role of DC in immune regulation by identifying signals that control the development of immunostimulatory DC. Pathogens, inflammation or immune cells activate the migration of tissue-resident DC to local lymphoid organs and induce their functional maturation for effective presentation of antigens to T lymphocytes [reviewed by (2,3)]. A pivotal consequence of such process is the production of DC cytokines which is dynamically modulated by the environment, including reciprocal interactions with T cells via CD40/CD40 ligand (CD40L) (4,5). The production of IL-12 by so-called type-1 DC leads to T cell responses with high levels of IFN-{gamma} and low levels of IL-4 or IL-10 (6). Maturation of DC also determines the outcome of antigen presentation by inducing high and specific levels of T cell-interacting molecules such as MHC and co-stimulatory antigens. The underlying molecular mechanisms engaged in DC that coordinate the features of activation remain incompletely characterized.

The study of DC transcription factors has provided valuable clues. NF{kappa}B becomes activated in murine and human DC after exposure to multiple pro-inflammatory signals including TNF-{alpha} and CD40L (79). Specific NF{kappa}B subunits, p50 and c-Rel, are required for the induction of IL-12 production and the survival of DC (10). In contrast, RelB is critical for expression of CD40, CD86 and MHC class II on DC during maturation (11). This suggests that a complex control of DC characteristics modulates T cell-mediated immune responses.

Specific transcriptional regulators, including NF{kappa}B constituents, also determine the existence of the various subsets of DC in murine lymphoid organs [(10,12) and some recently reviewed in (13)]. In particular, Ikaros family members regulate DC lineages in vivo in mice (14). Ikaros is the founding member of a family of Krüppel-type zinc finger transcriptional regulators comprising Ikaros, Aiolos, Helios, Eos and Pegasus (1517) that control the development of the hemato-lymphoid system [reviewed in (18)]. Multiple Ikaros isoforms exist that differ in the number of N-terminal zinc fingers enabling DNA binding. A mutant isoform lacking DNA-binding domains, IK7, can dimerize with other Ikaros isoforms or with family members via C-terminal interactions, thus functioning as a dominant negative regulator for this family of proteins (19). Mice expressing the IK7 dominant negative mutation are deficient in CD8{alpha}+ and CD8{alpha} DC and Ikaros null animals lack CD8{alpha} DC in lymphoid organs, thus demonstrating the importance of these factors in the development of DC lineages (14). Our own studies in human cells confirm the importance of this family of proteins for DC formation. Hematopoietic progenitor cells (HPC) expressing IK7 fail to differentiate in vitro into CD1a+ DC in response to specific cytokine signals (20). Such study also delineated differential needs for Ikaros proteins in various systems of human DC hematopoiesis. Indeed, IK7 does not block the differentiation of HPC into CD1a+ cells in response to GM-CSF, TNF-{alpha} and IL-4 cytokines, suggesting that the development of monocytic DC precursor cells, which respond to these cytokines (21), may be unaffected by IK7. Irrespective of effects on hematopoietic lineages, this system enables the production of human CD1a+ DC in the presence of IK7, providing a model to analyze the functional characteristics of DC with impaired activation of Ikaros family members. It is not known if Ikaros proteins can, similarly to NF{kappa}B, regulate any of the functional attributes of DC maturation.

This led us to investigate in greater detail the role played by Ikaros proteins in the later stages of human DC development. Using retroviral transduction, we over-expressed IK7 into human HPC and analyzed production of immunostimulatory APCs in particular via monocytic precursor cells. We report that functional Ikaros proteins are not required for monocyte-derived DC formation but are required for the acquisition of optimal type 1 APC function.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Human cells
Human cell samples were obtained with approval from the Institutional Review Board of Wayne State University and included human umbilical cord blood (UCB) specimens, human adult bone marrow (BM) cells isolated from ribs removed during thoracic surgery and apheresed mobilized peripheral blood (MPB) of breast cancer patients. From these tissues, mononuclear cells (MNC) were prepared by centrifugation over Ficoll (Pharmacia, Piscataway, NJ). Monocytes were obtained by plastic adherence of MPB MNC and monocyte-derived DC (Mo-DC) were prepared as described (22). BM or UCB CD34+ cells were purified by positive selection using an indirect method and goat anti-mouse magnetic beads (Miltenyi Biotech GmBH, Sunnyvale, CA) as described (23). Purified UCB naive T cells were obtained after removal of monocytes, B cells, phagocytes, APC and erythrocytes using negative selection with sheep anti-mouse magnetic beads (Dynal) and mAbs specific for CD14 (3C10–1E12), CD40 (G28.5), CD32 (IV-3), CD11b (OKM1), glycophorin A (10F7MN) (prepared from hybridomas obtained from ATCC, Manassas, VA), HLA-DP/DQ (SPVL-3) (kind gift of Dr H. Yssel) and CD16 (KD1) (kind gift of Dr G. Ferlazzo, Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy) and mAbs to HLA-DR (Caltag, Burlingame, CA). Two consecutive rounds of negative selection were performed yielding >95% pure CD3+ naive T cells.

Retroviral production and transduction
The construction of the bi-cistronic vectors LZRS-IK7-IRES-EGFP and its control LZRS-IRES-EGFP encoding murine Ik7 and enhanced green fluorescent protein (EGFP) or only EGFP have been reported elsewhere (20,23). Stable high-titer virus-producing Phoenix-amphotropic packaging cells (kind gift of Dr G. Nolan, Stanford University, Palo Alto, CA) were used to produce virus collected from confluent cell monolayers grown in R10 medium [RPMI-1640 medium (Gibco-BRL, Gaithersburg, MD) supplemented with L-glutamine (2 mM), penicillin–streptomycin (100U/ml and 100 µg/ml, respectively) and 10% fetal calf serum (Hyclone, logan, UT)]. Virus-containing medium was filtered (0.45 µm) and stored frozen at –80°C. Viral titers were determined by measuring EGFP expression after infection of HCT116 cells colon carcinoma cells. Virus batches of titer > 5 x 105 UEGFP/ml were used.

Transduction of CD34+ cells (5 x 105 cells/ml) was performed after 48 h pre-activation at 37°C in a humidified atmosphere with 5% CO2 in R10 medium supplemented with human recombinant c-kit ligand (50 ng/ml), interleukin-6 (25 ng/ml) and interleukin-3 (12.5 ng/ml). Cells were spin-infected (24) at 1000 g for 3 h at room temperature with 1 ml of virus supernatant in the presence of 20 mM Hepes in 24-well plates coated with 8–11 µg/cm2 fibronectin fragments (Retronectin, Takara Biomedicals, Shiga, Japan). After centrifugation, cells were incubated overnight at 37°C with fresh virus supernatant in the presence of the above-described cytokines. The next day, cells were washed and further cultured with fresh medium and cytokines.

Cell sorting and culture of transduced cells
Live transduced cell subsets expressing EGFP and excluding propidium iodide (PI) (Sigma, St Louis, MO, 5 µg/ml) were obtained by flow cytometry sorting (Vantage sorter, Becton Dickinson, San Jose, CA). When indicated, cells were stained either for CD34, CD1a or CD14 using phycoerythrin (PE)-conjugated anti-CD34 mAbs, PE anti-CD1a, allophycocyanin or Tricolor-conjugated anti-CD14 mAbs (Caltag, Burlingame, CA). Sorted cells (5–10 x 104/ml) were cultured in 24-well plates (Corning Costar Corp, Oneonta, NY) in appropriate medium. Differentiation of CD34+ hematopoietic progenitor cells into monocytes and/or DC was induced by culture with different combinations of cytokines described elsewhere (5,20): FKGm17: Flt3 ligand, c-Kit ligand, GM-CSF (25 ng/ml each, kind gift of Dr B. Hill, SyStemix Inc or of Immunex, Seattle, WA), IL-7 and IL-1ß (10 ng/ml each, R&D Systems, Minneapolis, MN) or FKGmT4: Flt3 ligand, c-Kit ligand, GM-CSF (25 ng/ml each), TNF{alpha} (50 ng/ml, RDI, Flanders, NJ or R&D Systems), and IL-4 (kind gift of Dr H. Yssel, DNAX, Palo Alto, CA) (100U/ml). Medium was changed by demi-depletion every 3 days.

Confocal microscopy
Cells spun on glass slides were air-dried and fixed in cold methanol, thus eliminating EGFP fluorescence of transduced cells. Cells were stained with FITC-conjugated anti-human HLA-DR (Caltag) and rabbit polyclonal anti-Ikaros antibodies [kind gift of Dr Katia Georgopoulos, Cutaneous Biology Research Center, Charlestown, MA, (19)] revealed by Texas Red-conjugated goat anti-rabbit antibodies (Molecular Probes, Eugene, OR). Negative controls consisted of cells stained with FITC-conjugated irrelevant mouse IgG, pre-immune rabbit serum and anti-rabbit conjugate. Cells were examined by confocal microscopy (Zeiss LSM 310) using a 488 nm Argon laser and a 63x objective.

Cell surface marker analysis by flow cytometry
Non-specific binding was blocked with human {gamma}-globin (Gamimune, Miles, Eckhart, IN) for 10 min followed by addition of mAbs such as: PE anti-CD1a, anti-CD54, anti-CD11c, anti-CD4 (Caltag), PE anti-CD83 (Caltag or PharMingen, San Diego, CA), PE-anti CD80 (Pharmingen), allophycocyanin anti-CD14 and allophycocyanin anti-MHC class II (Caltag). In some experiments, indirect stainings were also used to detect CD80, CD86 (Pharmingen), CD40 (G28.5) or MHC class I (W6/32) antigens using PE-conjugated goat anti-mouse Ig (Chemicon, Temecula, CA). Negative controls included IgG1 and IgG2a irrelevant mAbs. Data were acquired using a FacsCalibur instrument (Becton Dickinson) and analyzed with WinMDI software, version 2.8 (The Scripps Research Institute, La Jolla, CA).

MLR
Mixed lymphocyte reactions (MLR) were initiated by incubating allogeneic naive UCB T cells and APCs in R10 for 5–10 days. At indicated times, culture medium was collected and stored frozen at –20°C prior to analyzing cytokine content by ELISA with OPTEIA human INF{gamma} Kit, OPTEIA human IL-10 Kit (PharMingen, San Diego, CA) and IL-4 ELISA kit (R&D Systems) according to the manufacturer’s instructions. To measure T cell proliferation, APCs were irradiated (3000 cGy from a 131Cs source, JL Sheperd, San Fernando, CA), washed and added at various concentrations to 5 x 104 allogenic UCB T cells in 96-well U-bottom tissue culture plates (0.2 ml of R10 medium per well). Proliferation was measured after adding 1 µCi of 3H thymidine (DuPont NEN, Boston, MA) per well during the last 12 h of culture. Results were expressed as stimulation indices (SI) = average counts per minute of triplicate wells in test culture/average counts per minute of triplicate wells of T cells alone.

IL-12 p70 production
Cells were cultured with human recombinant CD40-ligand (1 µg/ml, kind gift of Immunex) in R10 medium for 24 h. Supernatant fluids were collected to measure IL-12 secretion by ELISA using the OPTEIA human IL-12 (p70) Kit (PharMingen, San Diego, CA according to the manufacturer’s instructions).


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Expression of Ikaros proteins in Mo-DC
Little is known of the expression of any Ikaros family member in human DC or their precursor cells. Using immunofluorescence and confocal microscopy we observed that Ikaros levels were below detection in human adherent blood monocytes (Fig. 1; day 0). However differentiation into Mo-DC was accompanied by induction of high levels of Ikaros in the form of speckles in the nucleus. Levels peaked between days 4 and 7 of culture, coinciding with optimal differentiation of Mo-DC (25). Levels of Ikaros subsequently declined in intensity but persisted for 2 weeks (Fig. 1). Thus, Ikaros proteins are present in Mo-DC and are up-regulated during their differentiation/maturation. Possibly, Ikaros proteins may play a role in this process.



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Fig. 1. Ikaros protein expression in Mo-DC. Adherent blood monocytes (day 0) were cultured with FKGmT4 cytokines and cells were sampled at different times of culture, spun on slides and fixed in cold methanol. All samples were stained for Ikaros protein expression using indirect immunofluorescence and analyzed simultaneously by confocal microscopy.

 
Expression of DN IK7 in CD34+ cells reduces the production of mature DC
To evaluate the importance of Ikaros proteins in the production of functional DC, we expressed the dominant negative mutant IK7 by retroviral-mediated gene transfer into multipotential HPC as described previously (20). Transduced EGFP+ CD34+ cells were purified by flow cytometry then subsequently cultured in the presence of cytokines flt-3-ligand, c-kit ligand, GM-CSF, TNF-{alpha} and IL-4 (FKGmT4) to induce DC differentiation and maturation as previously reported (5,20,25). Within a week, CD1a+ CD14 DC represented 23–69% of the cells in control cultures and 10–55% of cells in IK7 cultures (Table 1). Cells with typical dendritic morphology were seen in both cultures. Thus, we confirm that IK7 did not prevent the differentiation of CD34+ cells into CD1a+ cells in response to FKGmT4 cytokines (20), and extend these observations to show that CD1a+ CD14 DC expressing IK7 can be produced in this system.


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Table 1. Effects of IK7 on surface markers of HPC-derived cells
 
HPC-derived CD1a+ CD14 DC mature over time due to the presence of TNF-{alpha} in the culture medium, and express CD83, CD80, CD86, CD11c and HLA-DR antigens (25). In contrast, HPC expressing IK7 generated fewer cells expressing HLA-DR, CD83 and CD80 or CD11c (Table 1; whole cultures). Figure 2 illustrates a representative experiment. IK7 reduced intensity of HLA-DR, CD83, CD40, CD11c, CD80 and CD86 while CD1a was found on a large majority of the cells, not differently from control cultures. To facilitate the analysis of multiple markers on transduced DC, we purified EGFP+ CD1a+ CD14 DC by flow cytometry and after a short culture, cells were immunostained again (Table 1; purified DC). IK7 did not modify expression of CD80, CD86 or CD40, but the reduced expression of CD83 and HLA-DR was confirmed. This indicates that IK7 perturbed the production of mature DC from HPC and specifically affected the expression of HLA-DR and CD83 on the committed CD1a+ CD14 DC subset.



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Fig. 2. Flow cytometric analysis of HPC-derived cultures. Sorted EGFP+ CD34+ cells expressing or not the IK7 transgene were cultured for 8 days in the presence of FKGmT4 cytokines. Cell surface markers were analyzed by flow cytometry. Results representative of three experiments.

 
Expression of DN IK7 reduces the generation of type-1 DC activity
Type-1 DC can be produced in vitro by culture of human CD34+ HPC (21), notably in the presence of FKGmT4 cytokines as we reported (5,25). Indeed, activation of control HPC-derived cells with soluble trimeric CD40L induced the secretion of IL-12{alpha}ß in medium (Fig. 3). In contrast, IK7-expressing HPC-derived cultures secreted significantly less IL-12 (P = 0.003, t-test). T cell-activating properties of these APCs were analyzed in MLR. Control cells induced the proliferation (Fig. 4) and the production of IFN-{gamma} by naive allogeneic T cells (Table 2). Cells expressing IK7 also induced T cell proliferation, although a statistically-significant reduction (P < 0.01, t-test) was observed at the highest ratio of DC to T cell tested, but this was not apparent at other ratios. However, IK7-expressing APCs induced abnormal cytokine differentiation of T cells with lower production of IFN-{gamma} and higher levels of IL-4 than controls (Table 2; experiments 1 and 2). Altogether, results demonstrate that IK7 expression reduced the production of mature, functional type-1 DC from HPC.



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Fig. 3. IK7 reduced IL-12 production capacity in HPC-derived cells. Cells as in Fig. 2 were stimulated with CD40L overnight to measure IL-12{alpha}ß secretion in medium by ELISA. Results are expressed as pg/106 cells ± SD of three separate experiments.

 


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Fig. 4. Induction of T cell proliferation and differentiation in MLR. Representative MLR experiment out of two. Cells as in Fig. 2 were irradiated and used in MLR to stimulate cord blood purified T cells at various ratios of stimulator to responder cells. Proliferation was measured by thymidine incorporation at day 5.

 

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Table 2. Cytokine production in MLR
 
To better understand the effects of IK7 irrespective of possible effects on hematopoiesis, we purified CD1a+ CD14 DC from HPC-derived cultures. In MLR, such IK7+ CD1a+ CD14 DC consistently induced T cells to produce higher levels of IL-4 than control DC (Table 2; experiments 3–6). In contrast, the production of IFN-{gamma} was variable but overall not significantly modified from controls. Thus, IK7 expression in committed DC impaired antigen presenting cell function, reducing HLA-DR and CD83 expression and resulting in higher levels of IL-4 production by T cells.

IK7 does not prevent the formation of monocytes and Mo-DC
Next, we wanted to specifically examine the production and function of CD1a+ DC derived from monocytes as these are well-defined type-1 DC (26). First, we asked if this pathway of DC formation was affected by IK7. To generate Mo-DC from HPC, we established two-step cultures. First, HPC were cultured with the cytokines flt-3-ligand, c-kit ligand, GM-CSF, IL-1ß and IL-7 (FKGm17) to generate CD14+ cells (20). As seen in Fig. 5A (top panels), CD14+ cells were abundantly produced in control and IK7 cultures with no statistically significant difference in numbers of CD14+ cells produced (n = 5, data not shown). Subsequently, we added TNF-{alpha} and IL-4 to the cultures for 5 days to induce the differentiation and maturation of monocytic DC precursor cells into DC. Irrespective of IK7, this generated high proportions of CD1a+ CD14 DC (Fig. 5A, bottom panels, n = 2). Thus, IK7 did not prevent the production of monocytes from HPC and their subsequent differentiation into Mo-DC.



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Fig. 5. IK7 does not affect the formation of CD14+ monocytes or of Mo-DC but impairs Mo-DC cell surface marker expression. (A) Phenotype of cells in two-step cultures. Sorted EGFP+ CD34+ cells expressing or not the IK7 transgene were cultured in FKGm17 cytokines for 8 days and expression of CD1a and CD14 was analyzed on a fraction of cells (top panels). The remainder of the cells were then cultured with FKGmT4 cytokines for 5 days, then analyzed (bottom panels). (B–C) Phenotype of CD14+cell-derived DC. Transduced CD34+ cells expressing or not IK7 were cultured in FKGm17. After 7 days, CD14+ CD1a cells were sorted from these cultures and placed in FKGmT4 for 5 days (panel B) or 8 days (panel C) before flow cytometric analysis. Irrelevant staining controls (C) are shown.

 
IK7 affects T cell-stimulating ability of Mo-DC
Functional properties of Mo-DC obtained in two-step cultures were examined. In these experiments an additional control was included, consisting of cells exposed to the IK7 vector but failing to become infected. Such EGFP negative cells (IK7-neg cells) had a similar cell surface phenotype to control-transduced cells, displaying high levels of HLA-DR, CD83, CD11c, CD54 and CD80 (Fig. 5B and C). In contrast, IK7+ Mo-DC showed a marked down regulation of HLA-DR and CD83 with slight inhibition of CD11c and CD54 while CD80 expression was unaffected.

Mo-DC produce IL-12 in response to CD40L and the levels are regulated by temporal and maturation effects. IL-12 production capacity is highest in immature Mo-DC compared to DC previously matured with TNF-{alpha} (2729). The effects of IK7 were therefore tested in these two distinct stages of maturation. Immature DC were produced in two step cultures, respectively omitting or including the maturation agent TNF-{alpha}. Two experiments showed that IK7 did not significantly affect the production of CD40L-induced IL-12 by purified Mo-DC either in the immature or TNF-matured state (Fig. 6). Thus, IK7 caused abnormal phenotype in Mo-DC with reduced levels of HLA-DR and CD83 but IL-12 production seems unaffected in these cells.



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Fig. 6. IK7 does not affect IL-12 production capacity of Mo-DC. Immature or mature Mo-DC were produced in two-step cultures as in Fig. 5 except that CD14+ CD1a EGFP+ cells were cultured in FKGm4 (immature) or FKGmT4 (mature) cytokines for 5 days. DC were stimulated with CD40L to induce IL-12{alpha}ß secretion. Results are expressed as pg/106 cells ± SD of two separate experiments.

 

    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this study, we show that IK7 affects the phenotype and T cell-activating properties of committed DC. This demonstrates that normal levels of Ikaros proteins are required to control the late functional stages of DC development in addition to a previously recognized role in DC hematopoiesis (20).

We find that Ikaros family members play a role in committed DC produced in various systems of culture. A consistent effect of IK7 was the reduction of HLA-DR and CD83 on CD1a+ CD14 DC and Mo-DC. We also show that IK7+ DC derived from HPC reproducibly induced Th2 cytokines in T cells. Preliminary experiments seem to confirm these observations in Mo-DC because immature IK7+ Mo-DC induced higher levels of IL-4 and IL-10 in secondary MLR compared to control Mo-DC (data not shown). Th2 T cell development is known to be induced by low doses of antigen (3035). Being MHC class II low APCs, IK7+ DC would therefore present low amounts of antigen in MLR and thus could induce Th2 development through this mechanism. In uncharacteristic fashion, IK7+ DC concomitantly induced IFN-{gamma} with Th2 cytokines. This is probably due to their ability to produce IL-12 which is a dominant signal for the instruction of Th1 T cell differentiation (6,36). Thus, reduced HLA-DR expression with capacity to produce IL-12 is most likely responsible for the unusual co-induction of IFN-{gamma} and IL-4 by IK7+ DC. An interesting result is that IK7 profoundly affected the immune functions of APCs obtained in whole cultures. Contrary to purified DC, IK7 reduced not only HLA-DR and CD83 but also accessory and co-stimulatory molecules CD54, CD40, CD80, CD86 on the whole APC population. Hindering ICAM-1/LFA-1, CD40/CD154, CD80/CD28 interactions is expected to impair APC/T cell interactions and to bias the response towards Th2 T cell responses. Contrary to purified DC, IK7 also markedly reduced the capacity of the whole APC population to produce IL-12 which is expected to further impair Th1-inducing capacity (28). Indeed, we observed that whole APCs expressing IK7 induced less IFN-{gamma} and more IL-4 than control APCs. Intracellular T cell cytokine contents analyzed in one experiment, confirmed this trend and also showed the induction of distinct subsets of IFN-{gamma} and IL-4-producing T cells, suggesting that IK7-expressing APCs impaired Th1 responses while inducing Th2 T cell differentiation (data not shown). The observed phenotypic anomalies of IK7+ APCs may explain these findings but we cannot exclude that additional perturbations were also induced by IK7 including non-autonomous effects or generation of a distinct subset of APCs with specific Th2-inducing capacity. Since IK7 inhibited more IL-12 production in whole cultures than in purified DC, it is possible that IL-12-producing cells other than CD1a+ DC could be severely affected by IK7. Such cells were not studied here. We have recently shown that IL-12 production by human DC was critically dependent upon NF{kappa}B p50 (37). Preliminary results showed that IK7 reduced p50 expression in the HPC-derived cell population (data not shown) which is consistent with the observed effect on IL-12. This evokes complex interplays between the effectors of various signaling pathways in DC.

Our results also highlight that features of DC maturation, such as expression of HLA-DR and CD83, can be regulated independently of others. Mechanisms by which IK7 affects HLA-DR or CD83 are not known. We detected little intracellular HLA-DR in IK7-expressing cells (data not shown) excluding in principle an effect on retention of vesicular MHC class II complexes as found in immature DC. It is theoretically possible that Ikaros proteins regulate the transcription of MHC class II and CD83. Constitutive or inducible transcription of MHC Class II is regulated by RFX complex proteins that bind to class II promoter regions and recruit the MHC Class II transactivator CIITA (38). Ikaros core consensus binding sites (GGGAA) (39) were found in the 3' UTR of RFX-AP, in the 5' and 3' UTR of RFX-associated ankyrin containing protein, in the RFX2 and HLA-DR genes and more sparsely in the CIITA gene (A. Galy, unpublished observations) supporting this hypothesis. In addition, five core consensus binding sites were identified in the CD83 promoter (A. Galy, unpublished observations). Such speculations require testing in a separate study.

Thus, molecular mechanisms engaged during the process of DC activation are multifaceted and involve in part, Ikaros family members. At present it is not possible to implicate any specific member because the experimental system was non-discriminative, since IK7 associates and perturbs the activity of multiple partners within the family (19). Candidates include Ikaros itself which we find expressed in Mo-DC. We also detected Aiolos mRNA in Mo-DC and CD34+-derived DC (A. Galy, unpublished observations). Murine DC reportedly express Ikaros and Helios mRNA preferentially in splenic CD8{alpha}+ DC and in thymic DC whereas Aiolos is preferentially detected in splenic CD8{alpha} DC (15). The existence of multiple family members and the fact that DC development is more severely impaired in Ikaros DN mice than in Ikaros null mice (14) suggests that various aspects of DC formation, activation and maturation may require different Ikaros family members. Our results extend developmental studies in animals at several levels. In part, we find that normal activity of Ikaros proteins is not critical for the formation of human Mo-DC in vitro. Thus, specific DC precursor cells or specific signaling pathways in DC may be independent of Ikaros protein-mediated functions. By extrapolation to the murine system, this may explain why some populations of DC persist in the skin of DN mice while those in lymphoid organs are affected (14).

For the first time, Ikaros family members can be implicated in the functional activation of DC. Our data therefore provide a previously unrecognized element in the regulation of antigen presentation that should be considered in the signaling cascade involved in DC activation.


    Acknowledgements
 
We acknowledge the technical support of Amanda Labron and support of the Flow Cytometry Core Facility at the Karmanos Cancer Institute. We are thankful to Drs Nolan, Spits, Georgopoulos, Ferlazzo, and to Immunex, DNAX and SySTEMix Inc. for their gifts of reagents. Thanks to the staff in the Operating Room and in Pathology at Harper Hospital for their help in procurement of human rib marrow samples. This work was supported in part by a grant from the American Cancer Society RPG-98-183-01-CIM to A. Galy.


    Abbreviations
 
APC—antigen-presenting cell

BM—bone marrow

CD40L—CD40 ligand

DC—dendritic cell

DN—dominant negative

EGFP—enhanced green fluorescent protein

GM-CSF—granulocyte monocyte colony stimulating factor

HPC—hematopoietic progenitor cell

IL-12—interleukin 12

MLR—mixed lymphocyte reaction

MNC—mononuclear cell

Mo-DC—monocyte-derived DC

MPB—mobilized peripheral blood

PE—phycoerythrin

PI—propidium iodide

TNF-{alpha}—tumor necrosis factor alpha

UCB—umbilical cord blood


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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