IRF8 Governs Expression of Genes Involved in Innate and
Adaptive Immunity in Human and Mouse Germinal
Center B Cells
Dong-Mi Shin., Chang-Hoon Lee.¤, Herbert C. Morse III*
Laboratory of Immunopathology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, United States of America
IRF8 (Interferon Regulatory Factor 8) is a transcription factor expressed throughout B cell differentiation except for mature
plasma cells. Previous studies showed it is part of the transcriptional network governing B cell specification and
commitment in the bone marrow, regulates the distribution of mature B cells into the splenic follicular and marginal zone
compartments, and is expressed at highest levels in germinal center (GC) B cells. Here, we investigated the transcriptional
programs and signaling pathways affected by IRF8 in human and mouse GC B cells as defined by ChIP-chip analyses and
transcriptional profiling. We show that IRF8 binds a large number of genes by targeting two distinct motifs, half of which are
also targeted by PU.1. Over 70% of the binding sites localized to proximal and distal promoter regions with ,25% being
intragenic. There was significant enrichment among targeted genes for those involved in innate and adaptive immunity
with over 30% previously defined as interferon stimulated genes. We also showed that IRF8 target genes contributes to
multiple aspects of the biology of mature B cells including critical components of the molecular crosstalk among GC B cells,
T follicular helper cells, and follicular dendritic cells.
Citation: Shin D-M, Lee C-H, Morse HC III (2011) IRF8 Governs Expression of Genes Involved in Innate and Adaptive Immunity in Human and Mouse Germinal
Center B Cells. PLoS ONE 6(11): e27384. doi:10.1371/journal.pone.0027384
Editor: Michael B. Fessler, National Institute of Environmental Health Sciences, United States of America
Received September 2, 2011; Accepted October 14, 2011; Published November 11, 2011
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for
any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: This work was supported by the Intramural Research Program of the National Institutes of Health (NIH), National Institute of Allergy and Infectious
Diseases. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
. These authors contributed equally to this work.
¤ Current address: Center for Reproductive Medicine, Good Moonhwa Hospital, S&M Research Institute, S&M Incoporated, Busan, South Korea
IRF8, one of nine members of the IRF family of transcription
factors, functions in modulating immune responses and as a
central element in the IFN signaling cascade. The gene is
constitutively expressed in macrophages where it has been
identified at the promoter regions of a large number of genes
critical to macrophage differentiation and function [1–5].
Macrophages of mice deficient in IRF8 due to a conventional
gene knockout (KO)  or a spontaneous mutation (IRF8R294C) in
BXH2 mice  remain immature and are susceptible to a variety
of infectious agents [8–10].
Studies of IRF8-deficient mice also identified critical roles in
dendritic cell (DC) development and function. IRF8 KO mice lack
plasmacytoid DCs (pDC) and CD11c+CD8a+DCs [11,12];
however, R294C mutant mice lack only CD8a+DCs indicating
that distinct IRF8-dependent mechanisms mediate the develop-
ment of these two DC subsets.
Early on, it was shown that IRF8 is constitutively expressed by
normal mouse B cells and lymphoma cell lines with features of
pro-B and pre-B cells but not by plasmacytomas, tumors of mature
plasma cells . The contributions of IRF8 to early B cell
development in mice were found to include involvement in the
transcriptional networks controlling B cell lineage specification,
commitment and differentiation in bone marrow  with
regulation of the pre-B to B cell transition being dependent on
heterodimerization of IRF8 with another IRF family member,
IRF4 . The recent development of IRF8 conditional knockout
mice made it possible to determine B cell lineage-specific effects of
IRF8 deficiency . These studies showed that IRF8 normally
acts to control the sizes of both the splenic marginal zone and
follicular B cell populations while having little effect on responses
to immunization with T-dependent or T-independent antigens.
Additional studies showed that among mouse and human B
lineage cells IRF8 is expressed at the highest levels in germinal
center (GC) B cells and lymphomas of GC origin but is
extinguished in terminally differentiated plasma cells and plasma
cell neoplasms [17,18]. IRF8 was shown to contribute to the GC
reaction by modulating the expression of BCL6, AID and MDM2
[17,19]. Although some of the transcriptional programs and
cellular pathways that mediate IRF8 effects in myeloid and DCs
have been worked out in great detail [4,10,20,21], much less is
known about these aspects of IRF8 in B cell biology. The present
studies were directed at broadening our understandings of these
processes utilizing i) ChIP-chip analyses to identify IRF8 targets in
human and mouse lymphoma cell lines of GC origin, and ii) gene
expression profiling of a lymphoma cell line of GC origin and
IRF8 siRNA knockdown subclones.
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Results and Discussion
Identification of IRF8 targets in cell lines derived from
human lymphomas of GC origin
To identify direct transcriptional targets for IRF8 in human GC
B cells, we hybridized IRF8-bound chromatin obtained by ChIP
from three cell lines of GC origin (ODH1, VAL and LY1) to
Nimblegen promoter tiling arrays consisting of probes covering
3.5 kb upstream to 0.75 kb downstream of transcriptional start
sites (TSS); a multiple myeloma cell line (MMS1) with very little or
no expression of IRF8 served as a negative control. The number of
genes identified as IRF8-bound in the three GC lines were 1,563
for VAL, 1,724 for ODH1 and 2790 for LY1 with 271 genes being
common to all three lines (Figure 1A; Table S2). These binding
sites were identified by applying the false discovery rate
(FDR),0.01 to IRF8-specific enriched peaks detected by the
sliding window method.
Mapping of probes targeted by IRF8 to the human genome
showed that the great majority fell within well-defined peaks
located from 1 kb 39 to 1 kb 59 from the TSS of involved genes
(Figure 1B). In contrast no significant peaks were observed with
material prepared from the negative control cell line, MMS1,
although a low frequency of targets extended from 24 kb to
+1 kb. While target sites identified in ODH1 and VAL lying
outside this interval were indistinguishable from the pattern for
MMS1, a small subset of targets lying 2 kb to 3 kb upstream of the
TSS were seen for LY1 (Figure 1B).
An example of the fold enrichment of ChIP to input for each
cell line is shown in Figure 1C in relation to the TSS for TLR4
identifying a prominent peak directly over the TSS in all three
biological replicates (FDR,1E24) with no significant binding
seen with MMS1. We then used ChIP-qPCR to validate the
results of ChIP-chip binding assays for 15 genes identified in all
three cell lines as targets for IRF8 by ChIP-chip (Figure 2A).
Substantial enrichment was seen with most genes having at least
10-fold enrichment of IRF8 ChIP DNA compared to input DNA
with ChIP material from ODH1 and VAL. The same general
pattern was seen but with usually less enrichment with ChIP
material from LY1. The basis for this cell line-specific difference is
not understood. (Figure 2A, top). The heat map in the lower part
of Figure 2A showing fold enrichment of IRF8 ChIP to input
presented by ChIP-chip analysis demonstrated a high level of
correlation between data obtained by ChIP-qPCR and ChIP-chip.
An examination of the genes identified as having IRF8 binding
sites by ChIP-chip was performed by Gene Ontology (GO)
analysis and revealed significant enrichments for immune response
categories including innate and humoral responses, responses to
virus as well as antigen processing and presentation (Figure 2B).
The immune response category is comprised of 21 genes nearly
half of which encode proteins involved in antigen presentation by
MHC class I molecules (HLA–B, HLA–C, TAP1, TAP2, TAPBP,
PSMB8, PSMB9) or MHC class II molecules (HLA–DRA, CD74,
CIITA). Another large subset of genes encodes proteins involved
in anti-viral responses (OAS1, OAS3, MX2, IFI35, IFIT3, IFIT3,
Figure 1. Identification of IRF8 targets in human cell lines of GC B cell origin. Labeled IRF8 ChIP samples and input samples from the three
human cell lines of GC B cell origin were applied to Nimblegen HG18 385 k arrays and peak signals were analyzed by sliding window algorithm with
threshold of FDR,0.01. (A) Venn diagram of IRF8 targets in three cell lines. Numbers in parentheses indicate the numbers of targets identified in each
line. Internal numbers indicate targets common to two or all three lines. (B) Distribution of IRF8-bound sites relative to the transcription start site (TSS)
of target genes. (C) Representative IRF8 binding from ChIP-chip in four cell lines. Binding of IRF8 to the TLR4 promoter is shown as an example. Fold
change is calculated from relative fold enrichment of IRF8 ChIP signal to input signal. Dashed line shows TSS.
Transcriptional Network of IRF8 in B Cells
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Figure 2. Validation of IRF8 ChIP-chip and functional classification of IRF8 targets(A) ChIP-qPCR validation using primer pairs surrounding
the putative binding sites identified by ChIP-chip. For each locus, the fold enrichment comparing IRF8 ChIP DNA to input DNA is represented in the
bar graph. The heatmap (lower panel) shows fold enrichment obtained from ChIP-chip. (B) Categorization of IRF8 targets by Gene Ontology (GO).
Percents of genes in each category in the whole array or in the set of IRF8 targets are shown. p-values indicate significance of the enrichment for IRF8
targets in each GO category. (C) Motif analysis for IRF8 ChIP hits. Over-represented motifs were identified by TRAWLER and MEME. (D) A Venn diagram
of IRF8 targets and interferon-responsive genes. The Interferome DB was used for identifying interferon-responsive genes.
Transcriptional Network of IRF8 in B Cells
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IFIT5) or other aspects of IFN signaling (IRF9, BCAP31). The
overlap with GO descriptions identified in similar ChIP-chip
analyses of IRF8 target genes in myeloid cells is substantial , but
is clearly and predictably demarcated by the category of humoral
immune response. A seeming superimposition of B cell-specific
and AID-dependent receptor diversification on the substrate
provided by the classical innate immune functions of macrophages
is in keeping with the suggestion of an earlier appearance of B than
T cells in adaptive immunity, although other interpretations are
To identify the characteristics of the cis-regulatory motifs over-
represented in the set of IRF8-bound targets, repeat-masked ChIP
sequences were queried in TRAWLER . Two over-represent-
ed position weight matrices were generated by comparison to
human 1000 bp upstream genome sequences as a background
(Figure 2C). The top matrix (Z score=22.99) contains two tandem
canonical IRF binding sites (TTTC) separated by two nucleotides,
characteristic of the IRF9/STAT1/STAT2 binding site termed an
interferon stimulated response element (ISRE) . The bottom
matrix (Z score=19.78) contains an IRF target sequence
separated by two nucleotides from a TTCC motif that serves as
a binding sequence for ETS family members including PU.1
(SPI1) . This matrix closely resembles the previously identified
TTTCNNTTCC motif, designated an ETS-IRF composite
element (EICE) [1,26]. MEME (Multiple EM for Motif Elicitation)
is another widely used tool for searching for novel ‘signals’ in sets
of biological sequences leading to discovery of new transcription
factor binding sites. MEME analysis of the same data set identified
a matrix (p=1.1E–73) strikingly similar to the ISRE- and EICE-
like motifs identified by TRAWLER (Figure 2C). We conclude
that the DNA targets for IRF8 binding are divided among those
that require heterodimerization with other IRF family or ETS
family members. Although there are differences between the IRF8
binding sites identified by ChIP-chip in activated macrophages 
and those defined here, they are basically very similar, reinforcing
the concept of important commonalities between the transcrip-
tional programs of macrophages and B cells.
Both IFNa/b and IFNcinitiate transcriptional activation of
IFN-stimulated genes (ISGs) by activation of the JAK-STAT
signaling pathways [24,27,28]. This results in binding of STATs as
well as IRF and ETS family members to various IFN response
elements including ISREs and EICEs described above. To
examine the potential contributions of IRF8 to regulation of ISGs
in GC B cells, we determined the proportion of IRF8 target genes
that are part of the ‘‘Interferome’’ database of ISGs (http://www.
interferome.org/; ; 30.4% of the IRF8 targets overlapped with
the 1,996 genes in the Interferome database (Figure 2D, Table S2).
Taken together, the results of our ChIP-chip analyses of human
GC-derived lymphoma cell lines identified over 250 target genes
with binding sites located primarily at TSSs. The target sites were
highly enriched for two distinct binding motifs very similar to
canonical ISRE and EICE elements, respectively. A high
proportion of the target genes were included in the Interferome
of ISGs and were functionally involved in aspects of both innate
and acquired immunity including antigen processing and
Identification and characterization of IRF8 target sites in
mouse lymphoma cell lines of GC origin
Our initial impetus for studying possible contributions of IRF8
to B cell development and function came from analyses of mouse B
cell lineage lymphomas showing that levels of IRF8 expression
varied significantly at progressive stages of differentiation.
Expression was highest in diffuse large B cell lymphoma (DLBCL)
of GC origin but was almost totally absent in tumors of mature
plasma cells . In that study, the IRF8-expressing NFS-202 cell
line of GC B cell origin and IRF8 siRNA-expressing stable
transfectants of that line were examined for selected gene
expression by qPCR and for IRF8 target genes by ChIP. In the
present study, we extended these analyses by examining these
lines, two other IRF8-expressing cell lines of GC B cell origin
(NFS-201 and NFS-205) and the IRF8-negative plasmacytoma cell
line, MPC11, for gene expression profiling by microarray and for
IRF8 and PU.1 target screening by ChIP-chip.
ChIP-chip analyses identified 3,659 and 2,672 IRF8 binding
sites in NFS-201 and NFS-202, respectively, but only 1,290 sites in
NFS-205 (Figure 3A). Among the targets, 871 were found to be
common to all three lines (Table S3), a number 3.2-fold higher
than for targets common to the three human cell lines. The
reasons for this species-related difference are not clear but could be
explained if the mouse lines were more similar to one another in
differentiation state than the human lines or by the fact that all the
mouse lines derive from a common NFS genetic background .
The lower number of IRF8 target sites identified in the NFS-
205 cell line was also of interest. IRF8 differs from other members
of the IRF family in that it can bind DNA only after
heterodimerization with other members of the IRF family or with
non-IRF transcription factors such as PU.1 [31,32]. This
prompted us to determine if PU.1 was expressed at comparable
levels in the three cell lines. Unexpectedly, western blot analyses
revealed significant differences among the lines for PU.1
expression while IRF8 levels were relatively similar (Figure 3B).
PU.1 protein levels were high in NFS-201, substantially lower in
NFS-202 and below the limits of detection in NFS-205.
Mapping of probes targeted by IRF8 in the three cell lines to the
mouse genome paralleled studies of human IRF8 target sites with
the majority mapping within 1 kb upstream or downstream of the
TSSs of involved genes (Figure 3C). Interestingly, the proportion
of sites mapping to this region was considerably lower for NFS-205
than the other cell lines, raising the possibility that a significant
proportion of sites in the larger peaks documented for NFS-201
and NFS-202 may be targeted by IRF8/PU.1 heterodimers that
tend to result in promoter activation . In addition, a long
shoulder of target sites in all three lines mapped from 1 kb to
,4 kb 59 to the TSSs, proportionally more than was seen with the
human target sites. In contrast, no significant peaks were observed
anywhere throughout this region with material prepared from the
negative control cell line, MPC-11.
A more detailed characterization of the target sites for their
localization to proximal or distal promoters and to intragenic
regions is presented in Figure 3D. In the two PU.1-expressing cell
lines, there was an enrichment for targets in proximal as compared
to distal promoter regions (,40% vs. ,33%, respectively) with
another ,25% mapping as intragenic. Nearly 60% of the
intragenic sites were localized to 59 UTRs with almost none
mapping to 39 UTRs. Another third were intronic while less than
10% mapped to coding exons. Parallel studies of the PU.1-
negative NFS-205 cell line revealed a predictable enrichment for
targets mapping to distal promoter regions when compared to the
PU.1-positive cell lines without much change in the proportions
mapping to intergenic sites or their distributions among subsets of
these sites (Figure 3D).
These results indicated that genes targeted by IRF8 in germinal
center cells of both humans and mice are most often characterized
by binding sites in proximity to TSSs while also suggesting that the
differential distribution between proximal and distal promoter
regions is likely to be influenced by the availability of PU.1 as a
Transcriptional Network of IRF8 in B Cells
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Expression profiling of IRF8 regulated genes
To identify genes affected at the transcriptional level by
alterations in IRF8 expression, we performed gene expression
(SAM) tool, we identified 954 down-regulated genes and 1107 up-
regulated genes in IRF8 knockdown cells (Figure 4A, Table S4),
consistent with the activities of IRF8 as both a transcriptional
activator and a transcriptional repressor. To validate the transcrip-
tional effects of IRF8 suppression identified by microarray analyses,
we quantified expression of 29 affected genes that were present on a
commercial qPCR array. There were strong correlations between
the expression levels of genes determined by either approach (1/
slope =0.74, r2=0.90) (Figure 4B).
A gene ontology (GO) assessment of genes affected transcrip-
tionally by downregulation of Irf8 revealed enriched gene clusters
associated with a variety of cellular processes centered on
hematopoietic differentiation as well as cell-mediated and humoral
immune responses (Figure 4C, top panel). Molecular functions
affected most prominently by altered IRF8 expression were those
involved in cell development, growth and proliferation, but also
cell death and cell-to–cell signaling (Figure 4C, bottom panel).
We next applied Gene Set Enrichment Analysis (GSEA) to
determine if the expression of genes identified as targets of IRF8
binding by ChIP-chip was altered by suppressing IRF8 expression
in NFS-202 cells (Figure 4D). The results showed that IRF8 target
genes were significantly enriched in either up-regulated genes or
down-regulated genes in the IRF8 knock down cell line with the
Figure 3. Identification of IRF8 and PU.1 targets in mouse cell lines of GC B cell origin. Labeled IRF8 ChIP samples and input samples were
applied to Nimblegen MM8 385 k arrays and peak signals were identified by sliding window algorithm with threshold of FDR,0.01. (A) A Venn
diagram for IRF8 targets. Numbers in parentheses indicate the number of IRF8 targets identified in each line. Venn diagram shows the number of IRF8
targets that belong to each area. (B) Western blots for IRF8 and PU.1 in the three cell lines. (C) Distribution of IRF8 ChIP hits by chromosomal location
relative to transcription start sites (TSS). (D) Distribution of IRF8 binding related to the annotated structure of associated genes (top). The frequencies
of IRF8 hits in the sub-structure of intragenic locations (bottom).
Transcriptional Network of IRF8 in B Cells
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Figure 4. Transciptome analysis of mouse DLBCL cell lines stably expressing siIRF8. Total RNAs from NFS-202 cell lines stably expressing
siIRF8 and control cell lines were applied to NIAID mouse expression arrays. (A) Significant Analysis of Microarray (SAM) plots for identification of
differentially expressed genes in knock-down cell lines. Both up- and down-regulated genes were identified with FDR,0.01. (B) qPCR validation of
differentially expressed genes from microarray analysis. Fold change of siIRF8 cell lines vs. control cell lines were plotted against fold change in
microarray. Values are in log2 scale. Linear regression analysis was performed (p,0.0001). (C) Functional classification of significant genes in IRF8
knock-down cell lines. Fisher’s exact test was performed to identify significantly enriched biological categories using Ingenuity Pathway Analysis (IPA).
Log-transformed p-values from Fisher’s exact test are shown on the x-axis. (D) GSEA analysis of mRNA expression profiles for IRF8 knock-down cells
vs. control cells. Relative expression was rank-ordered by fold change of five replicate IRF8 knock-down samples vs. five replicate control samples.
Genes associated with IRF8 binding sites (IRF8 ChIP targets) were strongly correlated with IRF8 expression level. The color bar at the bottom indicates
up-regulated (red) and down-regulated (blue) genes. (E) Distribution of PU.1 ChIP hits by chromosomal location relative to transcription start sites
(TSS). PU.1 ChIP-chip analyses were done using NFS-201 and NFS-202 cells that express PU.1 and NFS-205 cells that are PU.1-negative. Labeled PU.1
ChIP samples and input samples were applied to Nimblegen MM8 385 k arrays and peak signals were analyzed by sliding window algorithm with
Transcriptional Network of IRF8 in B Cells
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bottom part of enrichment plot showing where IRF8 targets
appear in the ranked list of genes in the expression array.
Relationship of PU.1 binding to subsets of IRF8 target
PU.1 is a key transcription factor required for the development
of all hematopoietic cells , for lineage fate decisions leading to
B cell and macrophage differentiation , and for effector
functions of mature macrophages. Several recent studies are also
suggestive of roles for PU.1 in differential distribution of B cells
into the splenic follicular and marginal zone compartments 
and as a negative regulator of late B cell differentiation ,
although specific target genes have not been identified. Prior
studies demonstrated that both PU.1 and IRF8 are recruited to
DNA sequences defined as EIREs (ETS/IRF response elements)
or EICEs (ETS/IRF composite elements) that lead to transcrip-
tional activation [1,37]. The fact that the number of IRF8 target
sites was significantly reduced in PU.1-deficient NFS-205 cells
suggested that a significant number of germinal center B cell genes
may be targeted by PU.1/IRF8 heterocomplexes.
To examine this possibility, we first used ChIP-chip analyses to
characterize PU.1 target sites in NFS-201 and NFS-202 cells with
NFS-205 serving as a negative control and identified 1,764 target
sites common to both IRF8-expressing cell lines (Table S5). As for
IRF8 targeted locations, the great majority of target sites for both
lines mapped within 1 kb 59 to 1 kb 39 to the TSSs of involved
genes (Figure 4E). Interestingly, ,75% of the genes identified as
targets of PU.1 in GC B cells were distinct from those identified
previously by ChIP-chip analyses of PU.1 targets in the
macrophage cell line RAW264.7  (Figure S1). After eliminat-
ing false positive targets for IRF8, defined as those found in the
MPC-11 control cells, and those for PU.1, defined as targets
identified in the PU.1-negative NFS-205 cells, we identified 355
genes commonly targeted by both transcription factors.
By combining ChIP-chip and gene expression microarray
studies of the mouse cell lines, we identified 277 genes that were
targeted by IRF8 and that were significantly altered in expression
in siIRF8 knockdown cells (Figure 4F). The IRF8 targets were then
segregated into two clusters based on whether they were also
targeted by PU.1. The results of these studies revealed a near
50:50 split among IRF8 target genes for those that were also
targeted by PU.1 and those that were not. We then used the
Trawler algorithm to characterize binding motifs associated with
targets bound by IRF8 alone and those targeted by the presumed
heterodimers. Predictably, the canonical ISRE motif - GAAANN-
GAAA (TTTC A/G G/C TTTC) - was identified as the top
matrix in the subset of IRF8-only targets (Figure 4F; Z=11.4).
Similar analyses of the sites targeted by both IRF8 and PU.1
identified the sequence GTTTCACTTCC (GGAAGTGAAAC),
identical to EICE elements, as the most over-represented motif
A broader picture of the transcriptional landscape governed by
IRF8 in mouse B cells is presented in Table 1 in which IRF8 target
genes, categorized functionally, are further annotated for the
effects of IRF8-specific siRNA on gene expression and ChIP-chip
analysis of PU.1 binding. IRF8 target genes were associated with a
wide spectrum of biologic processes, including components of
innate and adaptive immunity, as highlighted previously for
human targets. In addition, substantial numbers of genes were
associated with the categories of GTP signaling, transcription
factors, cell adhesion, as well as secondary protein modifications
by ubiquitylation, SUMOylation and ADP ribosylation. ChIP-
chip studies showed that, as noted above, nearly half of the IRF
targets were also targeted by PU.1, and gene expression studies
indicated that IRF8 was directly involved in the expression of
,60% of the targeted genes. These results indicated that IRF8 is
involved in broader aspects of B cell biology than was appreciated
previously and that there is significant overlap between genes
regulated transcriptionally by IRF8 in B cells and those targeted in
macrophages or dendritic cells as reported by others [4,10,20,21].
IRF8 network common to human and mouse B cells
Comparisons of genome-wide transcription factor binding
patterns across species indicate that a large proportion of
enhancers are species specific with significant divergences between
human and mouse [39,40]. Our analyses of both mouse and
human cell lines of GC B cell origin allowed us to rephrase this
issue in terms of IRF8 targets in B cells. Using stringent criteria, we
identified 51 genes that were targeted by IRF8 in the cell lines of
both humans and mice, with further analyses demonstrating that
45% were also targeted by PU.1 (Figure 5). Among the 51 genes,
41 were represented on the expression arrays used for transcrip-
tional profiling of NFS-202 IRF8 knockdown cells, making it
possible to determine the relationships between target occupancy
and regulation of gene expression. Significant changes in transcript
levels detected for 27 of the 41 genes implied that 15 were
activated and 12 were repressed by IRF8 or IRF8 plus PU.1.
Functionally, over half of the targeted genes common to humans
and mice were readily identified as contributing to various aspects
of acquired and innate immunity with B cell signaling and
differentiation, antigen processing and presentation, anti-viral
activities and nucleic acid recognition being most prominent.
Importantly, 90% of these ‘‘immune’’ genes were previously
shown to be responsive to stimulation with type I, type II or type
III IFNs and, not infrequently, all three (Figure 5).
These observations prompted us to validate this network by
studying expression levels of MHC class II genes and CIITA in
B220 gated FAS+GL7+GC B cells of IRF8 conventional KO and
control mice. First, spleen cells from these mice were analyzed by
flow cytometry for the levels of the MHC class II expression on GC
B cells. Flow cytometric studies showed that the levels of MHCII
expressed by GC cells of IRF8 KO mice were significantly lower
than for cells of wt mice (MFI fold change =1.9, Figure 6A). We
also quantified transcript levels for C2ta, the master control factor
for expression of MHC class II genes and one of the class II genes,
H2-Ab1, following stimulation of WT and IRF8 KO B cells with
IFNc. The results showed that both genes were expressed at
The results of this study provide the first comprehensive picture
of the transcriptional programs and cellular pathways governed by
IRF8 in mature B lineage cells as viewed through the lenses
provided by analyses of cell lines of GC origin from humans and
mice. Our conclusions derive from a synthesis of data from ChIP-
chip analyses of IRF8 occupancy of target sites in both species,
threshold of FDR,0.01. (F) Classification of mouse IRF8 targets with altered expression by siIRF8 in relation to PU.1 binding to the gene. A Venn
diagram of genes bound by IRF8 from ChIP-chip and genes that were significantly altered in IRF8 knock-down cells as indicated by gene expression
microarray (false discovery rate,0.01). Those IRF8 targets with differential expression were classified into two groups based upon the observation of
PU.1 binding from ChIP-chip. The top over-represented motifs were identified in two groups of IRF8 ChIP-hits by TRAWLER using mouse 1 kb
promoter set as background.
Transcriptional Network of IRF8 in B Cells
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microarray-based transcriptional profiling of the mouse cell lines,
and ChIP-chip analyses of PU.1 target sites in the mouse cells. The
findings indicate that IRF8 is involved in the regulation of a large
number of genes of known importance to various aspects of the
biology of mature B cells.
As illustrated in Figure 7A, these targets include critical
components of the molecular crosstalk among the specialized cell
types that comprise the GC reaction – GC B cells, TFH, and
follicular dendritic cells (FDC). Communications between GC B
cells and TFHare mediated by a series of molecular pairings that
include the cytokine IL21 and its receptor, IL21R, the T cell
receptor and antigen presented my MHC Class II molecules,
CD40 and its ligand, CD40L, and PDL1 and its receptor, PD1.
These couplings promote enhanced secretion of IL21 by TFH,
driving the generation of both memory B cells and plasma cells
(reviewed in ). Brief encounters of antigen-specific B cells with
antigen present on the surface of FDC combine with survival
signals provided by TFH,through engagement of PDL1, by FDC
secretion of BAFF and APRIL, ligands for TACI, and CXCL13,
the ligand for CXCR5, and by pairing of Sonic hedgehog (SHH)
on FDC with Patched (PTCH) to promote positive selection,
affinity maturation and clonal expansion of GC B cells. Some of
the IRF8 target genes that either promote the expression and
activity of the B cell receptor/ligand pairs or contribute to their
downstream signaling pathways are listed under the different cell
Table 1. Functional and transcriptional features of IRF8 target genes in mouse cell lines of GC B cells.
ActivatedRepressed Not determined
B2m, C2ta, Erap1, H2-DMb1, H2-Ea,
H2-M3, H2-Q7, H2-Q8 Igl-V1, Rfx5,
Bcap31, Blnk, Btk, Cd274, Cd52, Cd74, ,
Ms4a1 (CD20), Sla
Cd40, Cd69, Dapp1, H2-D1, H2-DMb2, H2-K1, H2-K3,
H2-Q1, H2-Q5, H2-T10, H2-T17, H2-T22, H2-T9,
Innate immunityIfi35, Irf4, Mov10, Mx2, Zbp1Ifih1, Irf5, Isg20, Isgf3g, Ly86, Ncf4, Nosip,
Oas1c, Oasl2, Tlr4, Tlr9
Crry, Ddx58, Hmgb1, Ifit1, Igtp, Iigp2, Il12rb1, Irf2,
Irgm, Invs1abp, Oas1b, Tbk1, Tff1, Ticam2, Tlr12,
Tlr6, Trim21, Zc3hav1
Ccl5, Ccl6, Csprs, Epha2, Grina,
Il12rb1, Mst1, Ptch1, Socs1,
Aif1, Arnt, Btc, Ccrl2, Entpd1, Ltbp1,
S100a13, Spred2, Tbgr1, Tnfrsf13b,
Arts1, Blr1, Cxcl0, Il28ra, Phf11
DNA repair Gadd45g, Hus1, Parp9, Rad51l1, Shfm1 Bard1, Brca1, Ercc6l, Pold4, Xrcc5Dclre1c, Top3a, Trp53
ApoptosisApcs, Casp1, Cflar, Thyn1, Traf1Casp8ap2, Emp3, Pdcd11, Prdx6Bcl2a1a, Bcl2a1c, Bcl2a1d, Bid, Birc1f, Birc1g, Birc2,
Casp9, Tmbim1, Trp53
GTP signaling Cysltr2, Gem, Gnaz, Rabggta,
F2rl1, Gbp4, Igtp, Khdrbs1, Rab2b, Rabgap1l,
Arhgap25, Arhgap30, Fgd2, Gbp1, Gbp2, Gbp5,
Gimap9, Gnb2, Gnl31, Gpr18, Gprk5, Lsg1, Rab11b,
Rab21, Rab3ip, Rab8b, Rapgef6, Rkhd2, RP23-336J1.4,
Tagap, Tgpt, Ubxd5, Usp6nl
Phosphatase Ppm1k, Ptpn18, PtprcDusp2, Ppfibp2, Ppm1m, Ppp1r11, Ppp1r15b, Ptprj
Protein kinasesAkap13, Fgfr1op2, Stat1Aurkb, Madd, Miki, Skil, Spred2, Tbk1Btk, Csnk1a1, Flt3l, Prkrip1, Prkrir, Ptk2, Raf1, Socs1,
Stat2, Ttk, Ywhag
Cell cycle regulation,
Ccng2Cdkn2c, Cenpa, Mad1l1, Mad2l2, Tob2,
Cdca1, Rnf123, Tbrg1, Trp53
Eef1b2, Elf4, Mybl1, Nap1l3,
Armcx3, Atxn7l1, Bcl9l, Bcor, Cited2, Crem, Elf1,
Ets1, Irf7, Mnt, Mrcs2, Nfkb1, Nmi, Pcgf5,
Prrg2, Rnf141, Skil, Suv39h1, Zbtb32, Zfp422
Baz2b, Gtf3c5, Irf2, Irf5, Mzf1 (Zfp98), Rbbp9, Rfx4,
Rfx5, Sin3a, Sirt6, Tcea1, Tcof1, Usf1, Zfp143, Zfx,
Cbln3, Cd37, Epsti1, Itgb1,
Cd53, Lgals8, Lpxn, Mgat5, Pcdhgb4,
Cd164, Clec1a, Clec12a, Itgb3bp, Pdlim2
CytoskeletonClasp1, Elmo1, Exoc2, InadlEhd2, Ide, Katna1, Lsp1Clasp10, Marcks, Mast3, Ptk9l (Twf2), Tpt1, Tuba2,
Edem1, Nbr1, Psma2, Psmb10,
NcstnCtrl, Ctso, Ctss, Psme2, Uvrag
RNA processing, tran-
Cstf2t, Eif4a2, Erh, PrkrirCpsf2, Prkrip1, Qtrtd1, Sf3a3, Trove2,
Ddx21, Ell3, Pold4, Rnase4, Rexo4, Sf1, Tsen54, Xrn1,
Nedd4, Rnf123, Trim21, Ube1l,
Arl6ip1, Fbxo8, Mtbp, Parp8, Trim25,
Trim30, Uchl5, Usp18, Usp52, Zfp91
Fbxo17, Fbxo36, Fbxo39, Fbxo43, Parp14, Parp9,
Rnf31, Trim41, Ube2v1, Ufc1, Usp14, Usp44
Cct6b, Dnaja2, Dnajc10, Ppil3,
Arsk, Ebag9, Lrmp, Nvl2
Solute transporterOsbpl3 Cutc, Slc37a2
Mitochondria Cyp4v3, Fars2, Mrpl32Cap1, Mrpl3, Mrpl13, Oxsm
Fatty acid, cholesterol
Acad8, Plscr1, Stoml1
364 IRF8 targets with altered expression by siIRF8 were classified to their functional involvement in GC B cells (only known genes were listed). Genes in bold were
confirmed by qPCR; in italics are both IRF8/PU.1 targets.
Transcriptional Network of IRF8 in B Cells
PLoS ONE | www.plosone.org8 November 2011 | Volume 6 | Issue 11 | e27384
Transcriptional Network of IRF8 in B Cells
PLoS ONE | www.plosone.org9 November 2011 | Volume 6 | Issue 11 | e27384
Although not illustrated here, there are a large number of cell
membrane, cytosolic and endosomal proteins encoded by IRF8
targeted genes that function as sensors of pathogen-associated
molecular patterns. It is increasingly well recognized that when
engaged, these molecules can exert major influences on B cell
activation induced by BCR ligation or signaling through other
receptors [42,43,44,45]. While crosstalk between BCR and TLR
signaling thus contributes to normal responses to both T-
dependent and T-independent antigens, it is also clear that
aberrant activation of these signaling molecules can promote the
development of profound humoral autoimmunity [46,47,48].
Modulation of gene expression by IRF8 may thus contribute to
the balance between physiologic and pathoglogic B cell reactivity.
The molecular transitions required for the maturation of GC B
cells to plasma cells are governed by a relatively small set of
transcription factors that lie downstream of signals generated by
engagement of the IL21R, CD40 and the BCR (Figure 7B). B cell
identity is promoted by BCL6, PAX5 and BACH2, which
suppress transcription of PRDM1, in opposition to the drive for
plasma cell maturation advanced by XBP1 (not shown) and
PRDM1, which in turn suppresses PAX5 and BCL6. The
contributions of IRF4 to this scheme are complex and incom-
pletely understood as it is required for the differentiation and
function of mature B cells as well as plasma cells. The results of this
study showed that many of the components of this network are
transcriptional targets for IRF8 (Figure 7B).
Our systemic and comprehensive approaches have elucidated
the roles played by IRF8 in governing transcriptional network in
GC B cells. However, understanding the full nature of IRF8
contributions to B cell biology from the earliest stages of lineage
commitment to terminal differentiation will require more detailed
investigations of the partnering of IRF8 with other proteins at its
target sites. As noted previously, IRF8 can bind DNA only after
heterodimerization with other transcription factors. While our
studies demonstrated that IRF8 associates with PU.1 at about half
of the target sites defined in GC B cells, the full picture of IRF8
bound to these sites may be even more complex as IRF8 has been
shown to physically associate with both PU.1 and IRF4 to regulate
gene expression through recognition of ISRE and EICE sequence
Materials and Methods
Cell lines and mice
Human lymphoma cell lines of GC origin - LY1, ODH1, and
VAL - were kindly provided by Dr. Riccardo Dalla-Favera
Figure 6. MHC II expression in germinal center B cells of IRF8 knock out mice. (A) MHC class II expression in GC B cells in IRF8 KO and WT
mice. Representative histogram for MHC class II expression in GC B cells (left panel). Dot plot shows significant difference in MHCII expression
between two groups. *, p,0.05 from Mann-Whitney test. (B) MHC class II expression in in vitro stimulated B cells. Purified B cells were stimulated with
IFNc for 2days. Transcript level of Irf8 were determined by qPCR at days 1 and 2 after stimulation (left panel). Transcript levels of C2ta and H2-Ab1 in
wt and KO B cells determined by qPCR are shown (right panel). Data from four different mice in each group were examined by t-test. *, p,0.05.
Figure 5. IRF8 targets common in human and mouse. IRF8 targets common in both human and mouse were identified by intersecting human
and mouse ChIP-chip analysis. These 51 targets are listed along with data on fold enrichment of IRF8 ChIP vs. input. Data obtained from human cell
lines LY1, ODH1 and VAL are shown in the left most heatmap. IRF8 ChIP-chip data from mouse cell lines NFS-201, NFS-202 and NFS-205 are shown in
blue. Fold enrichment of PU.1 ChIP vs. input from NFS-201 and NFS-202 are shown in purple. IRF8 expression (Expr) column indicates expression
levels of genes identified as activated or repressed by IRF8. Reported responsiveness of genes to interferon type I, II, or III is shown in pale blue in the
rightmost map. Numbers in ChIP-chip data are fold enrichment and those in expression array are fold change of control vs. siIRF8 cell lines.
Transcriptional Network of IRF8 in B Cells
PLoS ONE | www.plosone.org10November 2011 | Volume 6 | Issue 11 | e27384
(Columbia University). LY1 and VALB are GC B cell type
DLBCL. ODH1 is a Burkitt lymphoma cell line of type I latency
for EBV infection. The MM cell line MMS1 was provided by
Dr. Michael Kuehl, National Cancer Institute, NIH and mouse
plasmacytomas cell line MPC-11 was from Dr. Michael Potter,
National Cancer Institute, NIH. Mouse cell lines of GC origin -
NFS-201, NFS-202 and NFS-205 - were generated in our
laboratory. NFS-201 and NFS-205 derived from DLBCL
tumors of centroblastic and immunoblastic subsets ,
respectively, and NFS-202 from the scid transplant of a
follicular B cell lymphma. NFS-202 IRF8 siRNA stable
transfectant was described previously . IRF8 KO mice 
and littermate controls were studied at six to ten wk of age.
Animal studies were performed under NIAID IACUC approved
Chromatin immunoprecipitation (ChIP)-chip
ChIP was performed according to the manufacturer’s protocol
(Nimblegen, Reykjavı ´k, Iceland). Briefly, cell lines were cross-linked
with formaldehyde and the chromatin extracts were sonicated
(Misonix Sonicater 3000). Following immunoprecipitation with anti-
IRF8 antibody (sc-6058x, Santa Cruz Biotechnology), DNAs were
purified from ChIP samples and input control samples. Purified
DNAs were blunt ended, ligated with linkers and amplified by PCR.
Amplified ChIP samples and input DNA samples were labeled with
Cy3 and Cy5, respectively. Labeled samples were pooled and
hybridized onto HG18 385 k two array set for human samples and
MM8 385 k two array set for mouse samples. ,60,000 transcripts
and ,48,000 transcript were represented in the human promoter
arrays and mouse promoter arrays, respectively.Arrays were scanned
(NimbleGene MS 200). Peaks were detected by searching for four or
Figure 7. The roles of IRF8 in GC B cells. IRF8 targets in B cells were presented in context of signaling pathways in GC.(A) Cross-talk between B
cells and T cells and follicular DC in GC. IRF8 targets in B cells are shown together with ligands or products from TFH or FDC cells. (B) Signaling
downstream of IL21R, CD40, and BCR. IRF8 targets are shown in red.
Transcriptional Network of IRF8 in B Cells
PLoS ONE | www.plosone.org 11November 2011 | Volume 6 | Issue 11 | e27384
more probes with signals above the cutoff value, which was a
hypothetical maximum, mean+6SD, using a 500 bp sliding window.
False discovery rate (FDR) score was calculated with 20 times
randomization (GSE30356 in GEO). ChIP targets were functionally
classified based on GO (http://geneontology.org) and the signifi-
cance of the enrichment for the ChIP targets in each category was
examined in GeneMerge (http://genemerge.cbcb.umd.edu).
Genomic sequences were retrieved from the UCSC databases
for HG18 and MM8. Over-represented motifs were analyzed
using Trawler in EMBL (http://ani.embl.de/trawler) and MEME
ChIP- Quantitative Real Time PCR (qPCR)
ChIP samples were also analyzed by qPCR. Primer designs
were based on IRF8 binding sequences from the ChIP-chip data;
primer sequences are listed in Table S1. The results are presented
as the fold-enrichment over input.
Total RNAs prepared from six replicate samples of NFS-202
cell lines stably transfected with IRF8 knockdown siRNAs 
and control vector transfected cell lines were applied to NIAID-
mouse gene expression arrays. Scanned images were analyzed as
detailed previously  and raw data were normalized with
LIMMA package in R (www.r-project.org). Differentially ex-
pressed genes were identified with SAM (Significance Analysis of
Microarrays; www-stat.stanford.edu/,tib/SAM) with 1% FDR.
Significant genes were divided into their functional categories and
tested for significance of those enrichments in IPA (Ingenuity
Pathway Analysis). The enrichment of IRF8 ChIP targets
identified by ChIP-chip in this expression profiling was also
examined by gene sequence enrichment analysis (GSEA) (http://
www.broadinstitute.org/gsea/index.jsp) (GSE30356 in GEO).
The methods used for qPCR were described previously .
RNA was prepared from cell lines and activated normal B cells
using the RNeasy mini kit (Qiagen) and the quality of the RNA
was examined by Bioanalyzer (Agilent). cDNA was synthesized
according to the manufacturer’s protocol (MessageSensor RT kit,
Ambion). cDNA was applied to 384-well mouse B cell qPCR
plates (Bar Harbor BioTechnology) and the reaction was carried
out using an ABI PRISM 7900 HT sequence detection system
(Applied Biosystems). All experiments were done in triplicate. The
correlations between gene expression as determined by microarray
and qPCR were tested with linear regression.
Western blotting was performed as described previously 
using antibodies to IRF8, PU.1 and b-actin (Santa Cruz).
Cells were prepared and stained as previously reported 
using monoclonal antibodies specific for B220, FAS, GL7, and
MHCII I-A(b) purchased from BD Pharmingen. Flow cytometric
analyses were performed on a FACSCalibur (BD). Data were
analyzed by FlowJo (BD).
B cell activation
Splenic B cells were purified with DynalBeads (Invitrogen) and
cultured at 16106/mL in 24 well plates with RPMI media
containing 10% FBS and 1% penicillin/streptomycin at 37uC.
Cells were activated with IFNc (500 ng/ml) for 2 days. Total
RNAs were extracted and transcript levels of MHCII and C2ta
were measured by qPCR.
PU.1 targets in macrophage cell lines. Comparison of PU.1
targets in B cell with PU.1 targets in macrophage cell lines
(GSE9011 in GEO). A Venn diagram (left) shows partial overlap
between B cell and macrophage targets in addition to B cell-
specific or macrophage-specific targets. Fold enrichment of PU.1
ChIP to input in B cells were plotted against fold enrichment
observed in macrophage ChIP-chip (right).
Comparison of PU.1 targets in B cell with
List of primers for ChIP-qPCR.
lines. FDR(false discovery rate) is ,0.05. Score is the fold
enrichment of ChIP to input in log2.
List of direct IRF8 targets in human GC B cell
lines. FDR(false discovery rate) is ,0.01. Score is the fold
enrichment of ChIP to input in log2.
List of direct IRF8 targets in mouse GC B cell
changes in their expression in IRF8 knock down cells.
1% of q-vaule(=FDR) in SAM was used to identify differentially
expressed genes in IRF8 knock down cells.
List of mouse IRF8 targets with significant
FDR(false discovery rate) is ,0.01. Score is the fold enrichment of
ChIP to input in log2.
List of PU.1 targets in mouse GC cell lines.
We thank Dr. Hongsheng Wang, Dr. Alexander Kovalchuk, Dr. Janet
Hartley and Dr. Ozato Keiko for many helpful discussions.
Conceived and designed the experiments: HCM CHL DMS. Performed
the experiments: CHL DMS. Analyzed the data: DMS HCM. Contributed
reagents/materials/analysis tools: HCM. Wrote the paper: DMS HCM.
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