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Departments of
* Immunology,
Neurology, and
Urology, Mayo Clinic College of Medicine, Mayo Clinic, Rochester, Minnesota, USA
1Correspondence: Department of Immunology, Mayo Clinic, College of Medicine, 200 First St. S.W., Rochester, MN 55905 USA. E-mail: pease.larry{at}mayo.edu
ABSTRACT
While bivalent antibodies can block ligand-receptor interactions, IgM pentamers efficiently cross-link cell surface targets and evoke physiological responses. We have described one such interaction between an IgM antibody (Ab) and the B7-DC costimulatory molecule expressed by dendritic cells that induces strong antitumor immunity and modulates pathogenic responses associated with allergic asthma. Progressive changes in gene expression in dendritic cells activated by an IgM B7-DC cross-linking Ab resulted in the increased expression in 350 genes and decreased expression of more than 200 genes over the course of 24 h following Ab treatment. In particular, up-regulation of the caspase inhibitor FLIP and the chemokine receptor CCR7, and the down-regulation of the CXCR4 receptor provide a mechanistic basis of Ab-induced survival and enhanced migration into draining lymph nodes. Increased expression of both cell surface and secreted molecules known to be mediators of the immunomodulatory properties of dendritic cells was detected at both the levels of RNA and protein expression. This analysis documents the ability of IgM Ab to activate a gene expression cascade leading to important biological changes in cellular function and provides mechanistic insight into the potent immunomodulatory properties attributed to this Ab.—Blocki, F. A., Radhakrishnan, S., Van Keulen, V. P., Heckman, K. L., Ciric, B., Howe, C. L., Rodriguez, M., Kwon, E., Pease, L. R. Induction of a gene expression program in dendritic cells with a cross-linking IgM antibody to the co-stimulatory molecule B7-DC.
Key Words: cellular activation • gene regulation • immune regulation • immune modulation • immunotherapy
A NOVEL APPROACH for altering cell function in vivo is the use of cell-specific pentameric IgM antibodies to initiate cell signaling by redistributing or aggregating cell surface membrane receptors. Our initial studies described this strategy in studies of Ab-induced repair of demyelination in mouse models of multiple sclerosis (1
2
3
4)
. More recently, we reported that another IgM Ab, sHIgM12, and its clonal version, rHIgM12 (collectively referred to as B7-DC XAb), can activate the immune system by engaging the membrane protein B7-DC (PD/L2) on the surface of dendritic cells (DC) (5
6
7)
. Because B7-DC is a known ligand for the PD-1 receptor on activated T cells, questions remain about the mechanism underlying immune activation by targeting B7-DC on the surface of DC. We favor the hypothesis that engaging B7-DC with the IgM Ab cross-links this cell surface protein, activating the antigen-presenting cells. In support of this view, DC treated in this way acquire, process, and present exogenous protein as antigenic peptides in an enhanced manner, migrate more efficiently into draining lymph nodes, live longer in a stressed environment, and display increased ability to activate naive T cells (5
6
7)
. Nonetheless, the mechanisms underlying the surprising physiological changes induced by treating DC with B7-DC XAb have not been firmly established.
It is important to understand mechanisms modulating DC function because these cells have the ability to alter the course of autoimmunity (8
9
10
11)
and can be used to develop cancer vaccines (12
13
14)
. DC have the capacity to shape the polarity of immune responses (15
16
17
18)
. Potent inflammatory cytokines, including TNF
and IFN
that support cellular immunity or cytokines such as interleukin (IL)-4 and IL-13, which support humoral responses are released differentially by T cells following modulation by DC. DC also may influence T cells to release tolerogenic cytokines such as IL-10 (19)
and TGFß (20
, 21)
, or they may induce tolerance by directly releasing IL-10 themselves (22)
.
Most approaches to activate DC employ agonists of the Toll-like receptor family or the TNF receptor family (23)
. These stimuli activate a maturation program that results in the down-regulation of antigen acquisition functions and the up-regulation of molecules, including costimulatory molecules of the B7 family and MHC class II antigen presenting molecules that provide important signals for the activation of naive T cells. We have reported previously that activation of DC with B7-DC XAb induces increased activation of antigen uptake and has no affect on the expression of either the B7 costimulatory molecules or class II MHC molecules. Therefore, the responses of DC to activation with B7-DC XAb or TLR/TNF receptor agonists appear to be quite different.
Despite these differences modulation of DC function in vivo with B7-DC XAb can have dramatically beneficial clinical effects. Systemic Ab treatment of animals bearing malignant melanoma spread throughout the lungs conferred strong antitumor immunity (24)
. Treatment of hyperimmune mice with the Ab reprogrammed the recall response to antigen and blocked the development of lung inflammation in a model of allergic asthma (25)
. B7-DC XAb activated bone marrow-derived DC mediate these same protective effects when adoptively transferred into hyperimmune mice (26)
, providing a convenient cell population for studies on the molecular mechanisms underlying Ab induced changes in cell function. Here, we report changes in gene expression in GM-CSF/interleukin-4-differentiated monocyte-derived DC induced by B7-DC XAb and delineate the cascade of changes in gene expression that regulate the striking biological consequences of DC activation stimulated by this IgM Ab.
MATERIALS AND METHODS
DC cultures
Immature DC from the bone marrow of C57BL6/J mice were generated with GM-CSF and IL-4 in culture using established procedures (6
, 27
, 28)
. Briefly, bone marrow was isolated from the long bones of the hind legs. Erythrocyte-depleted cells were plated at a density of 1 x 106/ml with 3 ml per well in six-well plates (BD BioSciences, San Jose, CA, USA) in RPMI 1640, supplemented with 10% cosmic calf serum (HyClone, Logan, UT, USA) + PSG, 10 ng/ml of murine GM-CSF and 1 ng/ml of murine IL-4 (PeproTech, Rocky Hill, NJ, USA). After 48 h incubation under 5% CO2 at 37°C, nonadherent cells were removed and media were replaced for an additional 5 d incubation.
Ab treatment
The human IgM Ab sHIgM12 was purified from the serum of a patient with Waldenstrom’s macroglobulinemia (6)
. The IgM antibodies used in these studies were enriched to greater than 95% purity, with only traces of other proteins visualized on SDS gel electrophoresis.
The B7-DC XAb sHIgM12 or polyclonal isotype-matched control human IgM Ab (10 µg/ml) were used to treat the 7 d bone marrow cultures. Two hours later, the B7-DC Ab-treated samples, PBS-treated, and isotype control Ab-treated wells were harvested using an RNA STAT-60 kit (Tel-Test, Inc. Friendswood, TX, USA) added directly to the adherent cells. The PBS and istoype-control samples displayed remarkably similar response patterns and served as the control group for the Affymetrix® gene chip studies. Twenty-four hours after Ab treatment, the remaining sHIgM12-treated wells were harvested in the same manner.
In experiments designed to determine the specificity of the Ab, binding to DC was critical for the observed responses; an IgM Ab that binds specifically to the MHC class II molecule IAb (25-9-3, BD Bioscience, San Jose, CA, USA) was substituted in the cell cultures for B7-DC XAb.
Real-time polymerase chain reaction (PCR) analysis
Bone marrow-derived murine DC (3x106) were cultured in GM-CSF and IL-4 for 7 d before stimulation with 10 µg/ml sHIgM12 or irrelevant sHIgm39 isotype control Ab for 30 min. After removing media from these adherent cells, lysis buffer was added and harvested lysate was homogenized using a QIA shredder column (Qiagen, Inc. Valencia, CA, USA). Total RNA was isolated using the Qiagen RNeasy kit. After quantification, the RNA were used in RT-PCR; Lightcycler and RNA Amplification Kit SYBR Green I Roche, ID Falls, ID, USA) as recommended by the manufacturer. Primers used were Egr-1, 5'-GCC GGA ACC CCA GAG G-3', and 5'-GGA GGC AAC CGA GTC GT-3'; c-Fos, 5'-AAC CGC ATG GAG TG-3', and 5'-GCT ACA GTA CGT GGA TAT AGC GAT T-3'; and c-Jun, 5'-CCA AGG GTA CAC AAG ATG-3' and 5'-AGA TCG AAT GTT AGG TCC-3'. Serial dilution of total RNA used for each reaction to determine the fold induction in comparisons for cross-linked samples with paired sham Ab-treated controls.
Transcript analysis by hybridization to microarray
Cells were harvested at 2 (control and sHIgM12) and 24 (sHIgM12 only) hours post-induction for total RNA using a RNA STAT-60 extraction kit. Total RNA samples were submitted to the Mayo Cancer Affymetrix® Core Facility for processing following Affymetrix® specifications (Santa Clara, CA, USA). The resulting biotin-labeled cRNA was hybridized on Affymetrix® Gene Chip Set MOE430A arrays to probes for 
23,000 transcripts and variants, representing over 20,000 mouse gene sequences. Hybridized gene chips were stained with streptavidin-phycoerythrin and scanned using an Affymetrix Gene Chip Scanner 3000 gene array scanner.
Microarray data analysis
Six paired treatment groups consisting of matched sham and B7-DC XAb- treated DC populations were compared, three at 2 h after Ab treatment and three at 24 h after treatment. Raw data from the mRNA expression profiles of all nine samples were culled using SpotfireTM Decision Site Software (Somerville, MA, USA) and queried populations downloaded to MicroSoftTM Excel spreadsheets for further analysis. The master Excel file for the murine universe registered 22,690 probes. This universe was culled by experimental groupings and purged of probes with no gene description or name [www.Affymetrix.com: Netaffx Analysis Center > Tools & Annotations > Expression > Batch Query (on probes)]. Sequential screening based on the strength and quality of probe hybridization [present, marginal, or absent] and the removal of replicate genes led to the production of unique gene working files.
Genes of known function were retrieved as follows. Initially, the 22,690 member parent murine probe universe was culled sequentially. First, probes that failed to exceed a 2-fold cut off value relative to its paired control sample were excluded. Only probes whose hybridization signal achieved a 2-fold increase or a 50% decrease in all three sample pairs at a given time point were considered further. Culled populations were parsed further of all probes that were designated "marginal" or "absent" using the Spotfire algorithm at the experimental time points for up-regulated genes or at the time zero control points for down-regulated genes. Probes with no gene name were also not considered to avoid possible duplications in probe sets representing unique genes exhibiting increased or decrease expression.
Antibodies used analytically
Commercially available antibodies used to assess the expression levels of selected proteins were anti ICAM-1 (YN1/1.7.4), anti 4–1BBL (TKS1), anti IL-6 (MF-2F3 and MP5–32D11, and anti TNF (TN3.19 and polyclonal) from eBioscience (San Diego, CA, USA); anti CXCR4 from BDPharmingen (San Diego, CA, USA); anti CCR7 (A19, affinity-purified goat) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); and anticFLIP (Dave-2) from Alexis (San Diego, CA, USA). Anti SLAM (D1) and anti-LY9 (30-C-7) monoclonal antibodies were gifts from Drs. William Faubion and David McKean of Mayo Clinic, respectively.
RESULTS
Our earlier studies indicated that B7-DC, a known ligand for the receptor PD-1 expressed by activated T cells, may itself be capable of back-signaling into DC (5
, 6)
. Using an adoptive transfer model, we demonstrated that the profound immunomodulatory effects induced by B7-DC XAb can be mediated by bone marrow-derived DC treated with Ab in vitro (26 and unpublished observations). To assess the direct effects of Ab treatment on DC function, we examine changes in gene expression profiles that are associated with these cultured DC treated with B7-DC XAb.
Induction of early response genes
In order to understand early molecular events induced by B7-DC XAb treatment of DC, we examined genes induced as early as 30 min after treatment. Immediate early genes (IEGs), including jun, fos, and egr family members, do not require protein synthesis for activation and are highly responsive to a variety of mitogenic and growth factor stimuli (29
, 30)
. IEGs promote a variety of functions specific to the type of cell in which they are induced (30
, 31)
. Within monocytic and dendritic cells, the transcription factor products of the genes c-jun, c-fos, and egr-1 induce expression of cytokines such as IL-6, IL-12, and TNF in response to signals mediated by MAPKs (32
33
34)
. Therefore, we asked whether the early phase of the DC response to B7-DC XAb induces IEGs. Real-time RT-PCR was used to compare levels of IEG mRNA from untreated DC against DC treated with B7-DC XAb for 30 min. A dilution series of RNA from sHIgM12-treated DCs revealed a 2.1-fold induction of c-fos mRNA, a 5.1-fold induction of c-jun mRNA, and a 7-fold induction of egr-1 mRNA over levels of corresponding genes in untreated DCs (Fig. 1
). Thus, the induction of IEGs may represent an early response pathway through which other downstream alterations in gene expression might occur in response to B7-DC XAb.
|
Profile of gene expression changes 2 and 24 h after treatment with B7-DC XAb
In order to assess the identity of genes being activated downstream of the IEGs in the treated DC, RNA was isolated from three independent paired-samples corresponding to control-treated and B7-DC cross-linked Ab-treated cells, 2 h after Ab treatment. Probes from the isolated mRNA molecules were prepared as described in Materials and Methods and used in hybridization studies using the Affymetrix gene chip platform and the MOE430A gene-chip array. The hybridization data were analyzed first by testing the null-hypothesis that variation of the hybridization signals detected in the analysis of paired comparisons between mRNAs from control DC vs. B7-DC cross-linked DC represented random fluctuations.
We tested this hypothesis by calculating the expected coincident frequency in three independent treatment groups showing a 2-fold increase in signal intensity with respect to their paired control. The probability that any paired comparison would equal or exceed a 2-fold increase was estimated from the observed frequency of such variants intrinsic to each of the three independent data sets. Given the three estimated frequencies, the probability that any random paired signal among more than 22,500 measured would be greater than 2-fold coincidently in all three data sets could be calculated as 4.6 x 10–4. Taking into account total probe sets assayed, the assumption of random fluxation in signal intensity led to the estimate of 10.4 probe sets in this category. However, 461 probe sets were actually found among the groups equaling or exceeding a 2-fold increase in the paired comparisons (P<0.001), indicating that activation of the DC with B7-DC XAb resulted in robust up-regulation of the mRNA encoded by a large set of genes (Table 1
). After the probe sets were culled as described in the Materials and Methods section, 229 distinct genes remained among the group of probes whose nonredundant genes exhibited a 2-fold or greater increase in mRNA following treatment with the B7-DC XAb. Using this same analytical strategy, 207 probes representing 89 known distinct genes were identified in all three samples displaying a 50% or greater decrease in expression 2 h after treatment, while only 18 probes were expected by chance to display 50% reduced hybridization.
|
In a parallel analysis of RNA molecules isolated 24 h after stimulation with B7-DC XAb, 15.3 probe-sets were expected to randomly fall within the group exhibiting a 2-fold increase or more, while 461 were detected, representing the presence of increased mRNA from 232 unique genes (Table 1)
. Remarkably, only 110 of these genes were represented in both the 2 and 24 h treatment groups (Fig. 2
). Even among the 110 shared genes up-regulated relative to matched controls at 2 and 24 h, considerable differences were apparent in expression levels at the two time points (Fig. 2)
, indicating that activation of immature DC with the B7-DC XAb sHIgM12 induces a progression of changes in gene expression. Similar to the gene expression pattern observed after 2 h, 196 under expressed genes were identified among 539 probes displaying 50% reduced expression, while only 16 were expected in this category (Table 1)
.
|
Independent validation of data interpretation
Our finding that 461 probe sets revealed a 2-fold or greater increase in signal intensity in RNA samples from DC activated with B7-DC XAb for 2 and 24 h, when only between 10 and 15 probe sets were expected, indicates that greater than 96 to 98% of the identified changes in accumulation of specific RNA molecules are likely to be actual changes. However, the status of particular gene products within this set can only be definitively ascertained by individual, independent assessment. A sample of 8 of the 351 distinct genes, whose mRNAs were found to be increased 2-fold within 24 h of DC treatment with B7-DC XAb, were chosen for evaluation to determine whether the increases in mRNA levels noted by the Affymetrix hybridization study could be validated at the level of protein expression. The genes were selected based on the availability of antibodies that could be used to detect the presence of the encoded gene products.
Clear associations between the chip assessments of increased mRNA levels and corresponding increases in protein expression were noted in each case after activation of DC with B7-DC XAb. Increased cell surface expression for the costimulatory family proteins 4–1BBL, SLAM, and LY9 and the adhesion molecule ICAM-1 was visualized using flow cytometry 6 h after DC activation (Fig. 3
). Increased expression of the cytokines TNF- and IL-6 was detected in culture supernatants by ELISA (Table 2
). Up-regulation of the chemokine receptor CCR7 (Fig. 4
A) and caspase inhibitor FLIP (Fig. 4B
) were detected by Western blotting. The set of gene products evaluated in this portion of the study is listed along with their corresponding estimates of increased mRNA levels in Table 2
. The finding that 8 of 8 gene products tested were in fact increased supports our conclusion that the vast majority of the gene products identified in our chip-based assay are actually increased in B7-DC XAb treated DC.
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Several subsets of genes whose expression appears to be elevated following treatment with B7-DC XAb are presented in the following tables: 8 genes pertinent to cytoskeletal rearrangement and endocytosis (Table 3
); 8 genes pertinent to migration (Table 4
); 12 genes pertinent to apoptosis (Table 5
); 10 chemokine family genes (Table 6
) and 28 cytokine family genes (Table 7
). In addition to the genes up-regulated following treatment with B7-DC XAb, a number of genes were also down-regulated, including a key chemokine receptor, signaling molecules, and transcription regulators (Table 8
).
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Specificity of DC activation following cell surface binding by IgM Ab
In the course of the previously described experiments, an isotype control Ab that does not bind DC appreciably was used to document Ab specificity. Using a wide variety of assays, we have consistently found that DC incubated with the irrelevant IgM Ab sHIgM39 behave the same as untreated DC. We take this as evidence supporting the hypothesis that the ability of B7-DC XAb to induce a response by DC is determined primarily by the IgM variable region and not by constant region interactions with determinants on DC, including Fc receptors. A second possibility is that constant region interactions only become important once an IgM Ab binds to the surface of DC. To evaluate this possibility, DC were incubated with an IgM Ab (25-9-3) that binds to MHC class II molecules and subsequently tested for changes in the expression of three proteins (LY9, ICAM-1, and SLAM) known to be up-regulated and one protein (CXCR4) representing the group of genes shown to be down-regulated following treatment with B7-DC XAb. As shown in Fig. 5
, DC bound by MHC class II-specific IgM Ab exhibited the same expression pattern for all four proteins observed in DC treated with the irrelevant isotype control Ab, while DC treated with B7-DC XAb exhibited up-regulation of LY9, ICAM-1, and SLAM, and down-regulation of CXCR4. These findings support our view that the activation of DC is mediated by the ability of B7-DC XAb to bind to its specific target on DC and that nonspecific Fc-mediated functions are not key for this Ab-induced response.
|
DISCUSSION
The human IgM Ab B7-DC XAb specifically induces in immature DC a progression of changes in gene expression that ultimately determines the ability of these cells to initiate and direct immune responses. Within minutes of cross-linking B7-DC, mRNA encoding the gene regulatory factors c-fos, c-jun, and egr-1 are induced. C-jun and c-fos (as well as other family members) can complex with one another to form the transcription factor activating protein (AP)-1 (activating protein-1) (34
, 35)
, a mediator of cell cycle control (36
37)
, as well as other functions. Cross-linking CD40 molecules on the surface of DCs enhances AP-1 binding to the IL-6 promoter (31)
. Stimulation of monocytes with lipopolysacharide results in binding of AP-1 to the TNF- and IL-12p40 promoters (32
, 33)
. Because TNF-, IL-6, and IL-12 transcripts are all elevated following Ab treatment of DC with B7-DC XA, signals derived from c-jun and c-fos formation of the AP-1 transcription factor might be involved.
In the course of these studies, we analyzed nine proteins to determine whether the observed changes in mRNA transcripts detected by the gene array studies were reflected at the protein level. All nine of the analyzed proteins were found at changed levels in DC following treatment with B7-DC XAb, providing definitive information validated by two independent methods demonstrating a subset of the responses by DC to this novel immune modulator. Three of these genes relate directly to physiological changes previously described in DC activated with B7-DC XAb. DC treated in vitro with B7-DC XAb migrate more efficiently to draining lymph nodes on adoptive transfer into the footpads of mice (5)
. The findings that the chemokine receptor CCR7 is up-regulated and CXCR4 is down-regulated in these DC are consistent with this functional change. These in vitro-treated DC also display enhanced survival in culture media devoid of the supporting cytokines GM-CSF and IL-4 (5)
. The increased expression of the caspase inhibitor FLIP is also consistent with the increased resistance to cytokine depravation, a strong apoptotic signal. The other six proteins analyzed also have potential significance to the immunomodulatory properties of B7-DC XAb, although the functional significance of each of these proteins has not yet been directly assessed.
One feature of the experimental design used in this study is that it allowed for the detection of small changes in gene expression (2- to 3-fold) in large sets of genes; some that represent newly expressed genes determining gains in function and some that encode elements of the cell’s regulatory machinery that are present at the time of activation and that are presumably consumed as a result of activation and subsequently replaced. While each has not been specifically validated in this report, we have provided a statistical argument that the regulation of most of these genes is likely altered following B7-DC XAb treatment of DC. Many of the genes for which increases or decreases of 2-fold or more were noted are known to play key roles in regulating immune responsiveness. Increased expression of the corresponding gene products would be consistent with the enhanced ability of the treated DC to activate a response from naive T cells (e.g., the increased expression of cytokines, chemokines, and costimulatory molecules such as IL23 (p19), RANTES, CXC11, CXC10, and CD40, see Tables 6
and 7
), altered migration patterns (up-regulation of chemokine receptor-like 2), and to tolerate the apoptotic signals prolonging the life of cells actively presenting antigen (increased expression of BclII, BclIII, and Serpinb9, see refs. 38
and 39 and Table 5
).
Analysis of another set of genes whose transcripts are present at the time of DC activation and displaying significant but modest changes within 2 h, may provide insights into the immediate signaling events that regulate the behavior of DC activation following cross-linking B7-DC. One large subset consists of genes associated with regulated changes in the cellular cytoskeleton (Tables 3
and 4)
. We have shown that DC treated with B7-DC XAb display increased ability to take up and process soluble antigen (5
, 24
, 28)
and migrate to draining lymph nodes (5)
, functions associated with cytoskeletal changes.
In addition to the up-regulated genes identified in this study, a group of down-regulated genes were also defined. The change in expression of one of these genes (CXCR4) was validated at the protein level. The identified genes provide a focused list of candidates for directing mechanistic studies aimed at understanding the ability of B7-DC XAb-activated DC to regulate the immune response.
An important implication of the finding that treatment of DC with B7-DC XAb results in gene expression changes is that cross-linking of B7-DC induces intracellular signals that influence gene transcription. In parallel studies, we have been investigating the nature of these signals. Among the genes found to be up-regulated by Ab treatment are a set known to be transcriptionally controlled by NF-
B. Recently, we have found that NF- B is activated following treatment with B7-DC XAb and that inhibition of NF- B activation results in the inhibition of IL-6 and TNF gene transcription (S. Radhakrishnan, L. T. Nguyen, B. Ciric, V. P. Van Keulen, M. Rodriguez, and L. R. Pease, unpublished observations). These studies delineating signaling pathways activated by B7-DC XAb treatment of DC provide independent confirmation of our conclusion that the Ab acts by directly activating DC. In the current study we have delineated a cascade of events leading to regulated increases and decreases in programs of gene expression.
ACKNOWLEDGMENTS
Supported by grants from the National Institutes of Health (grants R01 CA096859 and T32 AR007610) (L.R.P.) (grant R01 CA105138) (E. K.). The authors thank Rosalyn Cabrera for her contributions to Summary Fig. 1
.
Received for publication April 18, 2006. Accepted for publication June 19, 2006.
REFERENCES
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