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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. E-mail: pease.larry{at}mayo.edu
SPECIFIC AIMS
The human IgM antibody (Ab) B7-DC XAb is a potent immune modulator that activates antitumor immunity and can reprogram allergic antigen-specific immune responses preventing airway inflammation characteristic of asthma. Here, the effect of Ab treatment on gene expression in dendritic cells is evaluated, testing the hypothesis that cross-linking the costimulatory molecule B7-DC directly activates dendritic cells, modifying gene expression programs the control the functions of these central immune regulators.
Principal Findings
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 describing this strategy used IgM monoclonal antibody (mAb) to repair demyelinated lesions in mouse models of multiple sclerosis. Recently, a new IgM Ab, sHIgM12, and its clonal version, rHIgM12 (collectively referred to as B7-DC cross-linking antibodies, or B7-DC XAb), have been shown to activate dendritic cells (DC) by engaging the membrane costimulatory molecule B7-DC (alternatively known as PD/L2). DC activated 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.
Most approaches used to activate DC employ agonists of the toll-like receptor or TNF receptor families. 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. The 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.
Induction of early response genes
In order to understand early molecular events induced by B7-DC XAb treatment of DC, the expression of early-response genes induced as early as 30 min after Ab treatment were investigated using real-time polymerase chain reaction (RT-PCR). Immediate early genes, including the jun, fos, and egr transcription factor family members, do not require protein synthesis for activation and promote a variety of functions specific to the type of cell in which they are induced. Nevertheless, a 2.1-fold induction of c-fos, a 5.1-fold induction of c-jun, and a 7-fold induction of egr-1 mRNAs over levels of corresponding gene transcripts in untreated DCs was observed. Our interpretation is that treatment of DC with B7-DC XAb activates the transcription factors and that up-regulation of gene transcription occurs as homeostatic regulatory mechanisms come into play to restore the levels of these critical proteins.
Progressive changes in gene expression 2 and 24 h after treatment with B7-DC XAb
In order to assess the identity of genes being activated downstream of the immediate early genes in the treated DC, RNA was isolated from paired-samples corresponding to control-treated and B7-DC XAb-treated cells, 2 and 24 h after Ab treatment and used in hybridization studies using the MOE430A Affymetrix gene-chip platform. To identify changes in gene transcripts that were up- or down-regulated following Ab treatment, a simple, yet powerful, probability algorithm was applied to determine which probe sets were coordinately 2-fold higher or lower than control sample levels in three independent DC populations. As shown in Table 1
, 461 distinct probes were identified that exhibited a 2-fold or greater increase in mRNA hybridization levels in all three independent paired samples following treatment with the B7-DC XAb, whereas only 10 were predicted by chance alone. Using this set of probes, 229 corresponding genes were identified. Based on this same analytical strategy, 89 distinct genes were identified in all three samples displaying a 50% or greater decrease in expression 2 h after treatment; 232 unique genes were detected that were up-regulated 2-fold at 24 h and 196 that were down regulated 50% at this later time point. Remarkably, only 110 of these genes were represented in both the 2 h and 24 h up-regulated groups. Even among these 110 shared genes, considerable differences were found in expression levels at the two time points, indicating that activation of immature DC with the B7-DC XAb sHIgM12 induces a progression of changes in gene expression.
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Validation of the gene identification strategy
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 92 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 genes whose mRNAs were found to be increased 2-fold within 24 h of DC treatment with B7-DC XAb was 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. Clear associations between the assessments of mRNA levels and corresponding increases in protein expression were noted in each case after activation of DC with B7-DC XAb (Table 2
). Increased cell surface expression for the costimulatory family proteins 4–1BBL, SLAM, and LY9.2 and the adhesion molecule ICAM-1 was visualized using flow cytometry 6 h after DC activation. Increased expression of the cytokines TNF-
and interleukin (IL)-6 was detected in culture supernatants by ELISA. Up-regulation of CCR7, which governs skin DC migration under inflammatory and steady-state conditions, was detected by Western blotting. Finally, the increased expression of the antiapoptotic regulator FLIP in DC following treatment with cross-linking Ab indicates up-regulation of antiapoptotic signals in DC treated with B7-DC XAb. In a parallel study, the expression of the chemokine receptor CXCR4 was found to be decreased at both the level of RNA and protein expression. The finding that 9 of 9 gene products tested mirrored changes observed by RNA hybridization analysis supports our conclusion that the vast majority of the genes identified in our chip-based assay reflect actual increases and decreases in gene expression in B7-DC XAb treated DC.
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CONCLUSIONS AND SIGNIFICANCE
Activation of bone marrow derived DC in vitro with the B7-DC XAb induces a cascade of changes in gene expression beginning with the up-regulation of early response genes, then followed by changes in a wide spectrum of genes, many of which are known to perform specialized functions in the regulation of the immune response (Fig. 1
). 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 which are presumably consumed as a result of activation and subsequently replaced.
Statistical evidence, including the independent validation of 9 out of 9 genes tested at the protein level, suggests 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 TNF, IL23 (p19), Cxcl1, Cxcl10, RANTES, 4–1BB ligand, CD40, and ICAM1, to migrate into draining lymph nodes (increased expression of CCR7], and prolongation of the longevity of cells actively presenting antigen (increased expression of FLIP, BclII, BclIII, and Serpinb9). In addition a large subset of genes was identified that are known to regulate critical DC functions including phagocytosis, pinocytosis and cellular motility.
We have previously described the use of IgM antibodies to target cells in vivo for specific activation. The presumption has been that the targeted cells respond to this external stimulus by executing programmed genetic pathways. This study provides the first in-depth view of the consequences of activating a specific cell population using this strategy, revealing an extensive array of genes that are up- and down-regulated over a 24 h period following Ab treatment. Notably, many of the genes identified are known to determine the immunoregulatory properties of DC and provide candidate genes for delineating the mechanisms underlying the profound immunomodulatory events we have ascribed to this immunotherapeutic reagent (Fig. 1)
.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-6171fje
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