BACKGROUND
naïve mature b cells in peripheral lymphoid organs respond to a variety of extracellular signals through the activation of signal transduction pathways initiated by the b cell antigen, pattern-recognition, cytokine and chemokine receptors.
b cell responses to signaling depend on the combination of ligands present, and include activation, proliferation, migration, differentiation, isotype class switching, somatic hypermutation, anergy, and apoptosis  <cit> .
once activated, b cells can also serve as antigen presenting cells that preferentially present antigens recognized by their specific bcr.
in contrast, dendritic cells and macrophages present varied antigens that are acquired less specifically through phagocytosis, macropinocytosis and receptor-mediated endocytosis via pattern-recognition receptors such as the mannose receptor.
bcr-specific antigen processing and presentation is initiated by bcr-mediated signal transduction triggered by antigenic stimulation  <cit> .
antigen is then internalized by receptor-mediated endocytosis and trafficked through endosomes for acidification and fusion with lysosomes containing ph-sensitive hydrolytic enzymes for antigen processing.
endolysosomes containing processed antigenic peptides fuse with golgi-derived vesicles containing mhc class ii molecules assembled with invariant chain .
the clip fragment of ii bound in the cleft of the class ii aβ dimer is replaced by antigen-derived peptides and the complex trafficked to the cell surface through vesicle secretory pathways.
it is well known that b cell antigen processing and presentation mediated through the bcr far exceeds the efficiency of presentation of the same antigen by macrophages or dendritic cells  <cit> .
the mechanism giving rise to this increased efficiency has not been fully determined but appears to be a unique aspect of bcr-mediated antigen capture and processing as opposed to changes in the basic antigen processing and presentation machinery  <cit> .
one mechanism that may contribute to efficiency is accelerated trafficking of bcr/antigen complexes to class ii containing vesicles inside the cell  <cit> .
however, the molecular mediators of this vesicle trafficking, especially those components uniquely involved in the highly efficient b cell antigen processing and presentation pathway, have remained largely unknown.
we analyzed a b cell microarray dataset comparing the responses of normal splenic b cells to  <dig> individual ligands.
this study was designed to determine functionally important input signals to contribute to the understanding of normal b cell biology and differs from existing b cell microarray studies which largely compare normal b cells to b cell lymphomas to identify tumor-specific gene expression changes.
by combining this gene expression microarray analysis with bioinformatics analyses and functional assays, we have identified a set of specific genes that appear to be involved in bcr-mediated antigen capture, vesicle function and vesicle trafficking during b cell antigen processing and presentation.
these data provide a foundation for the molecular characterization of this important immunological process.
RESULTS
microarray analysis of ligand-treated b lymphocytes
purified b lymphocytes were treated in culture with  <dig> different ligands over a timecourse of  <dig> min,  <dig> hr,  <dig> hr, and  <dig> hr.
a detailed description of the data set has been published  <cit> .
following filtering, normalization and sam analysis, genes that were differentially expressed by each ligand in comparison with time-matched, untreated controls were identified.
of the  <dig> ligands, cd40l, lps, and aig caused the most gene expression changes, especially at the  <dig> hr timepoint .
further analysis focuses on these three immunologically-important ligands.
categorical values of  <dig>  - <dig>  and  <dig>  were used to group genes together based on their expression response patterns .
genes not differentially expressed under at least one treatment condition were omitted from further analysis.
based on  <dig> ligand combinations and a possibility of  <dig> outcomes for each ligand , there are a possible  <dig> or  <dig> gene clusters, with the  outcome excluded since only genes that were differentially expressed under at least one treatment condition were selected .
in our dataset, we only observe  <dig> of these possible  <dig>  a variety of different expression patterns were observed.
for example, gene cluster # <dig> contains genes that are upregulated by all three ligands, whereas gene cluster # <dig> contains genes that are only upregulated in response to lps.
classifi analysis links gene clusters to cellular physiology
microarray expression data can be full of experimental and biological noise, and many microarray probes are not well characterized.
we developed a gene cluster classification method that circumvents these limitations and links biological function with gene expression patterns derived from microarray experiments.
this method, termed classifi, uses gene ontology™ annotation to identify significant co-clustering of genes with similar biological properties, based on the postulate that genes involved in the same biological process would be coordinately expressed.
classifi utilizes the gene description database developed by the gene ontology™  consortium  <cit>  to reveal co-clustering of genes with similar biological properties.
the go annotates gene products with go terms based upon their molecular function, biological process, and cellular component.
these descriptive terms are structured in a directed acyclic graph of parent-child relationships, allowing a term to have multiple parents  <cit> .
go gene annotation has also been used by other groups as a tool for identifying biological processes relevant to gene expression profiling experiments  <cit> .
classifi measures the probability of co-clustering for every gene ontology term designated to every gene in each gene cluster.
gene expression data clustering and gene cluster membership assignment generates the classifi input file .
classifi captures all of the go terms annotated to every probe and then calculates the probability of chance co-clustering of go terms.
the final classifi output is a list of every go term, ranked within each gene cluster from lowest to highest probability .
the filtered data set contained  <dig> probes, which led to the calculation of  <dig> probabilities .
the go term with the lowest probability for each gene cluster is shown in table  <dig>  a significance cutoff of  <dig> × 10- <dig> was established using a bonferroni correction with an alpha of  <dig>  and an n of  <dig> .
six gene clusters give rise to go terms with significant probabilities: gene cluster # <dig>   <dig>   <dig>   <dig>   <dig>  and  <dig>  gene clusters identified by classifi to give significant probabilities exhibit predictable expression patterns that could be explained biologically, including gene clusters characterized by ligand-specific expression induction.
experimental noise may contribute to gene clusters with insignificant probabilities that show unusual expression patterns.
classifi results for some gene clusters are expected based on our current understanding of b cell physiology.
for example, the go term giving the lowest probability in gene cluster # <dig> is "nucleus".
this gene cluster represents genes which are upregulated in response to all three ligands: aig, cd40l, and lps, and includes genes involved in transcription, replication and rna processing.
all three of these ligands induce cellular activation  <cit>  and proliferation , so we would expect these kinds of nuclear genes to be highly expressed in b cells stimulated with all three ligands.
the go term giving the lowest probability in gene cluster # <dig> is "oxidoreductase activity, acting on nadh or nadph".
it has recently been shown that stimulation through cd <dig> and other tnf family members results in the production of reactive oxygen species through nadph  <cit> .
importantly, many of the go terms giving the low probabilities in a given gene cluster are functionally related, e.g.
monovalent inorganic ion transport, ion transport, transporter activity, and cation transport in gene cluster # <dig> .
experimental validation of classifi-derived biological predictions
classifi analysis should be viewed as a hypothesis-generating tool.
while the statistical analysis is compelling, predictions that derive from the hypotheses should be verified experimentally.
we were particularly interested in the aig-specific gene cluster # <dig> because these genes represent biological processes that are unique to antigenic stimulation rather than the general activation responses induced by all three ligands.
the go term giving the lowest probability in gene cluster # <dig> is "monovalent inorganic cation transport" .
an examination of the genes annotated with this go term revealed multiple components of the vacuolar atpase h+ pump .
since stimulation through the bcr induces internalization of the receptor-antigen complex and trafficking through the endocytic system, "monovalent inorganic cation transport" could reflect the acidification of vesicles as they progress from endosomes to lysosomes during endocytosis.
this aig-specific cluster also contained an overrepresentation of genes involved in general "transporter activity" , which includes genes like rab <dig>  sorting nexin  <dig>  and n-ethylmaleimide sensitive fusion protein attachment protein alpha  that are involved in endosome trafficking and vesicle fusion.
these observations led us to consider the hypothesis that aig might induce the expression of various components of the endocytic and vesicle transport pathways in a manner that is independent of its effects on the general metabolic activation and proliferation of b cells, and that this might be part of a b cell-specific antigen processing and presentation function.
to ensure that the genes identified through the classifi analysis were meaningful, we first sought to verify their differential expression patterns.
the microarray expression pattern of nine probes recognizing "transporter activity" genes in unstimulated and ligand-stimulated samples were evaluated .
in every case, expression of these genes was similar in all treatment conditions at  <dig>  and  <dig> hr, but was higher in the aig-treated samples at  <dig> hr and  <dig> hr.
examples in which the same gene is represented by two different probes show closely similar expression patterns, indicating the high quality of this microarray data set.
to verify the ligand-specificity of expression, rt-pcr was used to examine the expression of a subset of these genes in independently-generated samples.
again, aig induced enhanced expression of each of these genes compared to lps and anti-cd <dig> .
rt-pcr results consistently confirm microarray expression data in this data set  <cit> .
classifi analysis is based on the postulate that genes involved in the same biological process are coordinately expressed.
thus, one prediction of this analysis is that other genes involved in the same biological process that were not included in the microarray probe data set would show the same expression pattern.
we evaluated  <dig> such genes that encode atpase h+ pump complex subunits by rt-pcr.
all of these genes demonstrated a preferential upregulation in response to aig .
these data not only provide experimental validation of the classifi approach to microarray data analysis, but also further supports the hypothesis that aig induces vesicle processing and transport as a biological process.
in order to further test predictions that follow from the classifi analysis we sought to determine if the process of endocytosis is activated in a ligand/receptor-specific manner.
an internalization assay was performed in which the bcr is fluorescently labeled with a non-stimulating anti-igm-fitc antibody prior to aig treatment.
after allowing time for internalization, the cells are subjected to an acid wash to remove surface-bound label.
label that has been internalized is protected from the acid wash and can therefore be detected using flow cytometry.
using this method, we found that bcr internalization  occurs with aig stimulation, but not cd40l or lps stimulation in wehi- <dig> cells .
substantial internalization was found as early as  <dig> minutes post-stimulation and continued past  <dig> hours.
using fluorescence microscopy, internalized bcr can be seen in aig-treated cells by  <dig> minutes.
no internalization of the bcr is detected with anti-cd <dig> or lps treatment .
ligand-specific internalization of the bcr was also detected in primary b cells .
to test whether receptor internalization following aig stimulation is specific to the bcr and not other cell surface receptors, we performed internalization assays in which tlr <dig>  or cd <dig> was labeled prior to aig treatment.
in both cases, no internalization of the labeled receptor was detected , demonstrating that aig induces specific internalization of the bcr.
finally, to test whether stimulation of tlr <dig> with lps or cd <dig> with anti-cd <dig> induces tlr <dig> or cd <dig> internalization, we fluorescently labeled tlr <dig> or cd <dig>  then treated cells with the corresponding ligands.
in the case of tlr <dig> stimulation with lps, no internalization is detected .
anti-cd <dig> stimulation induces a rapid receptor shedding phenomenon  <cit>  and so it is difficult to directly compare the results of these samples with the other treatment conditions.
however, no evidence for internalization was found .
thus, aig induces a ligand-specific, receptor-specific endocytic process that is likely designed to capture specific antigen for processing and presentation.
finally, mhc class ii expression would be expected to be high in b cells undergoing antigen processing and presentation.
we found that following overnight stimulation with anti-igm, cd40l, and lps, class ii expression on anti-igm treated cells is sustained, while in cd40l and lps treated cells class ii expression is significantly downregulated .
this ligand-specificity for maintenance of class ii on the surface of anti-igm-treated cells is interesting because it suggests that b cells constitutively express a high level of class ii on the cell surface in anticipation of antigen presentation, which is then down-regulated in stimulated cells unless they are stimulated through the bcr.
this idea that b cells are primed to undergo antigen processing and presentation supports observations in the literature that antigen processing and presentation is accelerated with bcr stimulation.
further, our preliminary data indicate that b cells show enhanced stimulation of antigen-specific t cell clones in response to bcr stimulation .
discussion
classifi analysis
in this paper we report the analysis of gene expression responses of b lymphocytes to a panel of extracellular ligands using classifi, a statistical approach that uses go annotation to identify significant co-clustering of related genes, thus linking biological function with gene expression patterns derived from microarray experiments.
classifi circumvents two traditional limitations associated with microarray expression analysis – the effects of experimental and biological noise, and the variable depth of knowledge of individual genes in the probe set.
a well-characterized gene has relatively precise annotations that reach deep into the go hierarchy, while a poorly-characterized gene is only annotated with high level terms.
by capturing the entire ontology parentage of each gene, classifi overcomes limitations associated with variable depth-of-knowledge associated with most gene sets.
in addition, the probability calculation used in classifi is relatively robust to the addition of experimental noise, allowing the use of less stringent data filtering approaches, which reduces the false negative discovery rate without the usual associated increase in false positives.
indeed, increasing the sam fdr followed by classifi analysis increase the number of endocytosis-related genes while maintaining similar gene cluster classifications .
our analysis focused on the aig-specific gene cluster # <dig>  which was statistically classified as an intracellular transport-related gene cluster.
inspection of the entire gene list leads to the hypothesis that gene cluster # <dig> classification reflects the broader biological process of bcr-mediated endocytosis, vesicle acidification and trafficking, and antigen processing and presentation.
classifi should be considered a hypothesis-generating tool that requires experimental validation.
several predictions of our hypothesis were confirmed experimentally, including ligand-specific endocytosis induction and mhc class ii expression.
it should be noted that although we have used gene ontology annotation as a means of gene classification, any gene description scheme of interest could be used with the classifi approach to link experimental variables with legacy knowledge through gene expression microarray data.
identification of putative components of bcr-mediated antigen capture, processing, and presentation
based on the genes responsible for gene cluster # <dig> classification , we hypothesized that bcr engagement activates receptor-mediated endocytosis, vesicle acidification, vesicle trafficking, and antigen processing and presentation.
we manually curated the aig-specific cluster gene list and found a total of  <dig> genes, including those identified using classifi, that might be involved based on the molecular functions defined in other cell systems and biological processes from the literature .
figure  <dig> shows a schematic representation of the b cell antigen processing and presentation pathway indicating the putative roles played by these genes.
several genes known to be involved in signal transduction were found in gene cluster #18; some have been demonstrated to play a role in bcr signaling while others are involved in signaling pathways in other cell types and are candidates for new bcr signaling components that stimulate the endocytic process .
activation of protein kinases, including members of the map kinase family, in response to bcr engagement is well documented; thus strap, map4k <dig>  and map2k <dig> may also play a role in bcr signaling.
indeed, map4k <dig> is expressed in hematopoietic cells and is required for activation-induced t cell death following tcr stimulation, and map2k <dig> is required for il- <dig> production by macrophages and dendritic cells, and antigen-mediated activation of t cells.
cd <dig> has been found to regulate the cellular response to bcr signaling either through the recruitment of the shp- <dig> phosphatase thereby dampening bcr signaling, or by cooperating with bcr signaling to prevent apoptosis in immature b cells.
the dusp <dig> dual specificity phosphatase has been found to associate with the cd <dig> co-receptor and dampen tcr-mediated signaling in t cells by inactivation of map kinases, suggesting that it may regulate bcr signaling in a similar fashion.
daam <dig> is involved in wnt signaling in xenopus embryogenesis.
strap binds to the tgf beta type i receptor and inhibits downstream signaling.
whether these proteins might play a role in regulating bcr signaling remains to be determined.
the nf-atc <dig> isoform has been found to participate in the bcr-induced apoptosis in b-cell-derived burkitt's lymphoma cell lines, thus the presence of nfatc2ip and fkbp <dig> suggests that these interacting proteins may also be important in regulating ca++/calmodulin/calcineurin/nfat signaling in b cells.
a large proportion of genes in gene cluster # <dig> encode proteins with roles in endocytosis, vesicle trafficking, vesicle acidification, molecular processing and protein secretion.
in most cases these roles have been defined in other cell systems.
since little is known about the molecular details of these processes in receptor-mediated antigen capture and mhc class ii-dependent antigen processing and presentation in b cells, the possibility that gene cluster # <dig> has produced a putative list of proteins that coordinate these activities is intriguing.
snx <dig>  a member of the sorting nexin family, has been found to interact with clathrin in skeletal muscle.
clathrin has been implicated in facilitating membrane invagination during receptor-mediated endocytosis in b cells.
the presence of snx <dig> in this cluster suggests that it may help mediate this process.
the als <dig> protein acts as a guanine nucleotide exchange factor for rab <dig> and rac <dig>  and stimulates early endosome fusion.
rab proteins are small gtpases with homology to ras that play key roles in vesicle transport: rab <dig> has been found to localize near the apical and basolateral plasma membrane in epithelial cells of the kidney and intestine and rab22a has been found to localize to the plasma membrane and early endosomes.
iqgap <dig> is a scaffolding protein with multiple protein-protein interaction domains and a gtpase activation protein  domain that stimulates the hydrolytic activity of ras family members.
iqgap <dig> has also been found to localize to areas of membrane ruffling and newly formed vesicles in astrocytoma cells.
genes encoding homologs to several components of the vesicle-type proton pump were found in gene cluster # <dig>  including atp6v0b, atp6v1g <dig>  atp6v1c <dig>  and atp6v1f and the proton pump accessory protein atp6ap <dig>  additional proton pump components were also found to be induced in an aig-specific manner by rt-pcr .
together with the vesicle-specific chloride channel clcn <dig>  these proteins are likely to play a role in the acidification of the endocytic vesicles in preparation for their fusion with lysosomes to facilitate antigen hydrolysis.
proton pump components can serve as a membrane anchor point for snare proteins.
napa appears to mediate the tethering of these vesicles in preparation for fusion.
rab <dig> also appears to play a role in vesicle fusion since mutations in rab <dig> lead to the accumulation of lipid-rich vesicles.
several proteins involved in lysosome formation and antigen processing were found in gene cluster # <dig>  dysbindin binds to dystobrevins and is a component of the biogenesis of lysosome-related organelles complex  <dig> in mouse liver, which regulates trafficking to lysosomal organelles.
mutations in the cln <dig> gene leads to a lysosomal storage disorder associated with the accumulation of lipopigment-laden vesicles.
hexosaminidase a is a ph-sensitive hydrolase.
a variety of cathepsins are involved in mhc class ii antigen processing and presentation.
none of the probes for these genes passed our data filtering process, however the cathepsin inhibitor cystatin b was found in gene cluster # <dig>  suggesting that it might play a role in regulating cleavage site selection by modifying the relative activities of proteases involved in antigen processing and peptide loading.
hspbp <dig> may facilitate antigen processing by regulating the chaperone function of hsp <dig>  which has been associated with the presentation of myelin basic protein though mhc class ii in multiple sclerosis.
several genes involved in mhc class ii expression were found in the aig-specific gene cluster.
atf <dig> is a transcription factor that activates the promoter of the class ii trans-activator .
translocation of nascent mhc class ii peptide chains into the lumen of the endoplasmic reticulum involves the function of the sec <dig> translocation complex, the signal sequence receptor ssr <dig>  and the signal peptidase spc <dig>  calmegin  is a ca++ binding chaperone protein with significant homology to calreticulin and calnexin, an er chaperone protein that regulates the assembly of mhc class ii with the ii invariant chain.
vesicles containing peptide-loaded mhc class ii are transported to the plasma membrane for fusion and exposure on the cell surface, perhaps by traveling back through the tgn into the secretory pathway.
the vsp <dig> protein has been found to facilitate this kind of retrograde tgn transport in yeast.
in neurons, reticulon  <dig> is associated with synaptophysin in tubulovesicular structures and may play a role in the process of vesicle secretion.
cd <dig>  an mhc class ii co-receptor, may play a role in regulating mhc receptor compartmentalization.
<dig> genes found in gene cluster # <dig> encode proteins that have some connection with signaling, antigen capture, vesicle transport, vesicle acidification or mhc class ii expression, suggesting that activation of b cells through the bcr is inducing the expression of genes involved in mhc class ii antigen processing and presentation.
the activation of class ii presentation by antigen receptor engagement in b cells has been described extensively.
the findings reported here are novel in that microarray results combined with classifi analysis have provided a list of genes that may be involved in this important immunological process.
finally, it is intriguing to note the presence of genes involved in the regulation of apoptosis and autoimmunity  in gene cluster # <dig>  given the known function of the bcr in negative selection and the induction of autoimmunity.
transcriptional positive feedback
the changes in mrna levels for these genes involved in vesicle trafficking might be necessary to stimulate this biological process in aig-stimulated b cells.
however, the kinetics of the mrna changes compared with the rapid induction of endocytosis suggests that this may not be the case.
elevated mrna levels for these genes was found at  <dig> and  <dig> hr, but not at  <dig>  or  <dig> hr post-stimulation.
however, bcr endocytosis was maximally stimulated by  <dig> minutes.
the rapid induction of endocytosis suggests that at least the initiation of this process is activated through post-transcriptional mechanisms.
this finding suggests that the aig-specific transcriptional reprogramming observed in response to bcr stimulation may reflect positive feedback regulation in which expression of proteins that have already been activated is increased either as a means to amplify or sustain the process.
perhaps components of this important biological process are present at low levels in resting cells, and levels increase in response to appropriate environmental cues.
this kind of regulatory process might allow cells to respond rapidly to a broad set of variables in their changing environment while conserving energy and materials while in a resting state.
CONCLUSIONS
in this paper, we describe a bioinformatics analysis of a b cell microarray dataset using classifi to determine the biological significance of microarray gene clusters defined by ligand-specific b cell responses.
importantly, results from laboratory experimentation support the hypotheses derived from this microarray data mining exercise.
this work is a model for immunologists and other biologists for utilizing the growing field of bioinformatics for microarray data mining, hypothesis generation and hypothesis testing.
furthermore, the results from this analysis provide a foundation for a more detailed understanding of the b cell antigen processing and presentation pathway.

 naïve mature b cells in peripheral lymphoid organs respond to a variety of extracellular signals through the activation of signal transduction pathways initiated by the b cell antigen, pattern-recognition, cytokine and chemokine receptors. b cell responses to signaling depend on the combination of ligands present, and include activation, proliferation, migration, differentiation, isotype class switching, somatic hypermutation, anergy, and apoptosis. once activated, b cells can also serve as antigen presenting cells that preferentially present antigens recognized by their specific bcr. in contrast, dendritic cells and macrophages present varied antigens that are acquired less specifically through phagocytosis, macropinocytosis and receptor-mediated endocytosis via pattern-recognition receptors such as the mannose receptor. bcr-specific antigen processing and presentation is initiated by bcr-mediated signal transduction triggered by antigenic stimulation. antigen is then internalized by receptor-mediated endocytosis and trafficked through endosomes for acidification and fusion with lysosomes containing ph-sensitive hydrolytic enzymes for antigen processing. endolysosomes containing processed antigenic peptides fuse with golgi-derived vesicles containing mhc class ii molecules assembled with invariant chain. the clip fragment of ii bound in the cleft of the class ii aβ dimer is replaced by antigen-derived peptides and the complex trafficked to the cell surface through vesicle secretory pathways. it is well known that b cell antigen processing and presentation mediated through the bcr far exceeds the efficiency of presentation of the same antigen by macrophages or dendritic cells. the mechanism giving rise to this increased efficiency has not been fully determined but appears to be a unique aspect of bcr-mediated antigen capture and processing as opposed to changes in the basic antigen processing and presentation machinery. one mechanism that may contribute to efficiency is accelerated trafficking of bcr/antigen complexes to class ii containing vesicles inside the cell. however, the molecular mediators of this vesicle trafficking, especially those components uniquely involved in the highly efficient b cell antigen processing and presentation pathway, have remained largely unknown. we analyzed a b cell microarray dataset comparing the responses of normal splenic b cells to individual ligands. this study was designed to determine functionally important input signals to contribute to the understanding of normal b cell biology and differs from existing b cell microarray studies which largely compare normal b cells to b cell lymphomas to identify tumor-specific gene expression changes. by combining this gene expression microarray analysis with bioinformatics analyses and functional assays, we have identified a set of specific genes that appear to be involved in bcr-mediated antigen capture, vesicle function and vesicle trafficking during b cell antigen processing and presentation. these data provide a foundation for the molecular characterization of this important immunological process.  microarray analysis of ligand-treated b lymphocytes purified b lymphocytes were treated in culture with different ligands over a timecourse of min, hr, hr, and hr. a detailed description of the data set has been published. following filtering, normalization and sam analysis, genes that were differentially expressed by each ligand in comparison with time-matched, untreated controls were identified. of the ligands, cd40l, lps, and aig caused the most gene expression changes, especially at the hr timepoint. further analysis focuses on these three immunologically-important ligands. categorical values of - and were used to group genes together based on their expression response patterns. genes not differentially expressed under at least one treatment condition were omitted from further analysis. based on ligand combinations and a possibility of outcomes for each ligand, there are a possible or gene clusters, with the outcome excluded since only genes that were differentially expressed under at least one treatment condition were selected. in our dataset, we only observe of these possible a variety of different expression patterns were observed. for example, gene cluster # contains genes that are upregulated by all three ligands, whereas gene cluster # contains genes that are only upregulated in response to lps. classifi analysis links gene clusters to cellular physiology microarray expression data can be full of experimental and biological noise, and many microarray probes are not well characterized. we developed a gene cluster classification method that circumvents these limitations and links biological function with gene expression patterns derived from microarray experiments. this method, termed classifi, uses gene ontology™ annotation to identify significant co-clustering of genes with similar biological properties, based on the postulate that genes involved in the same biological process would be coordinately expressed. classifi utilizes the gene description database developed by the gene ontology™ consortium to reveal co-clustering of genes with similar biological properties. the go annotates gene products with go terms based upon their molecular function, biological process, and cellular component. these descriptive terms are structured in a directed acyclic graph of parent-child relationships, allowing a term to have multiple parents. go gene annotation has also been used by other groups as a tool for identifying biological processes relevant to gene expression profiling experiments. classifi measures the probability of co-clustering for every gene ontology term designated to every gene in each gene cluster. gene expression data clustering and gene cluster membership assignment generates the classifi input file. classifi captures all of the go terms annotated to every probe and then calculates the probability of chance co-clustering of go terms. the final classifi output is a list of every go term, ranked within each gene cluster from lowest to highest probability. the filtered data set contained probes, which led to the calculation of probabilities. the go term with the lowest probability for each gene cluster is shown in table a significance cutoff of × 10- was established using a bonferroni correction with an alpha of and an n of. six gene clusters give rise to go terms with significant probabilities: gene cluster # and gene clusters identified by classifi to give significant probabilities exhibit predictable expression patterns that could be explained biologically, including gene clusters characterized by ligand-specific expression induction. experimental noise may contribute to gene clusters with insignificant probabilities that show unusual expression patterns. classifi results for some gene clusters are expected based on our current understanding of b cell physiology. for example, the go term giving the lowest probability in gene cluster # is "nucleus". this gene cluster represents genes which are upregulated in response to all three ligands: aig, cd40l, and lps, and includes genes involved in transcription, replication and rna processing. all three of these ligands induce cellular activation and proliferation, so we would expect these kinds of nuclear genes to be highly expressed in b cells stimulated with all three ligands. the go term giving the lowest probability in gene cluster # is "oxidoreductase activity, acting on nadh or nadph". it has recently been shown that stimulation through cd and other tnf family members results in the production of reactive oxygen species through nadph. importantly, many of the go terms giving the low probabilities in a given gene cluster are functionally related, e.g. monovalent inorganic ion transport, ion transport, transporter activity, and cation transport in gene cluster #. experimental validation of classifi-derived biological predictions classifi analysis should be viewed as a hypothesis-generating tool. while the statistical analysis is compelling, predictions that derive from the hypotheses should be verified experimentally. we were particularly interested in the aig-specific gene cluster # because these genes represent biological processes that are unique to antigenic stimulation rather than the general activation responses induced by all three ligands. the go term giving the lowest probability in gene cluster # is "monovalent inorganic cation transport". an examination of the genes annotated with this go term revealed multiple components of the vacuolar atpase h+ pump. since stimulation through the bcr induces internalization of the receptor-antigen complex and trafficking through the endocytic system, "monovalent inorganic cation transport" could reflect the acidification of vesicles as they progress from endosomes to lysosomes during endocytosis. this aig-specific cluster also contained an overrepresentation of genes involved in general "transporter activity", which includes genes like rab sorting nexin and n-ethylmaleimide sensitive fusion protein attachment protein alpha that are involved in endosome trafficking and vesicle fusion. these observations led us to consider the hypothesis that aig might induce the expression of various components of the endocytic and vesicle transport pathways in a manner that is independent of its effects on the general metabolic activation and proliferation of b cells, and that this might be part of a b cell-specific antigen processing and presentation function. to ensure that the genes identified through the classifi analysis were meaningful, we first sought to verify their differential expression patterns. the microarray expression pattern of nine probes recognizing "transporter activity" genes in unstimulated and ligand-stimulated samples were evaluated. in every case, expression of these genes was similar in all treatment conditions at and hr, but was higher in the aig-treated samples at hr and hr. examples in which the same gene is represented by two different probes show closely similar expression patterns, indicating the high quality of this microarray data set. to verify the ligand-specificity of expression, rt-pcr was used to examine the expression of a subset of these genes in independently-generated samples. again, aig induced enhanced expression of each of these genes compared to lps and anti-cd. rt-pcr results consistently confirm microarray expression data in this data set. classifi analysis is based on the postulate that genes involved in the same biological process are coordinately expressed. thus, one prediction of this analysis is that other genes involved in the same biological process that were not included in the microarray probe data set would show the same expression pattern. we evaluated such genes that encode atpase h+ pump complex subunits by rt-pcr. all of these genes demonstrated a preferential upregulation in response to aig. these data not only provide experimental validation of the classifi approach to microarray data analysis, but also further supports the hypothesis that aig induces vesicle processing and transport as a biological process. in order to further test predictions that follow from the classifi analysis we sought to determine if the process of endocytosis is activated in a ligand/receptor-specific manner. an internalization assay was performed in which the bcr is fluorescently labeled with a non-stimulating anti-igm-fitc antibody prior to aig treatment. after allowing time for internalization, the cells are subjected to an acid wash to remove surface-bound label. label that has been internalized is protected from the acid wash and can therefore be detected using flow cytometry. using this method, we found that bcr internalization occurs with aig stimulation, but not cd40l or lps stimulation in wehi- cells. substantial internalization was found as early as minutes post-stimulation and continued past hours. using fluorescence microscopy, internalized bcr can be seen in aig-treated cells by minutes. no internalization of the bcr is detected with anti-cd or lps treatment. ligand-specific internalization of the bcr was also detected in primary b cells. to test whether receptor internalization following aig stimulation is specific to the bcr and not other cell surface receptors, we performed internalization assays in which tlr or cd was labeled prior to aig treatment. in both cases, no internalization of the labeled receptor was detected, demonstrating that aig induces specific internalization of the bcr. finally, to test whether stimulation of tlr with lps or cd with anti-cd induces tlr or cd internalization, we fluorescently labeled tlr or cd then treated cells with the corresponding ligands. in the case of tlr stimulation with lps, no internalization is detected. anti-cd stimulation induces a rapid receptor shedding phenomenon and so it is difficult to directly compare the results of these samples with the other treatment conditions. however, no evidence for internalization was found. thus, aig induces a ligand-specific, receptor-specific endocytic process that is likely designed to capture specific antigen for processing and presentation. finally, mhc class ii expression would be expected to be high in b cells undergoing antigen processing and presentation. we found that following overnight stimulation with anti-igm, cd40l, and lps, class ii expression on anti-igm treated cells is sustained, while in cd40l and lps treated cells class ii expression is significantly downregulated. this ligand-specificity for maintenance of class ii on the surface of anti-igm-treated cells is interesting because it suggests that b cells constitutively express a high level of class ii on the cell surface in anticipation of antigen presentation, which is then down-regulated in stimulated cells unless they are stimulated through the bcr. this idea that b cells are primed to undergo antigen processing and presentation supports observations in the literature that antigen processing and presentation is accelerated with bcr stimulation. further, our preliminary data indicate that b cells show enhanced stimulation of antigen-specific t cell clones in response to bcr stimulation. discussion classifi analysis in this paper we report the analysis of gene expression responses of b lymphocytes to a panel of extracellular ligands using classifi, a statistical approach that uses go annotation to identify significant co-clustering of related genes, thus linking biological function with gene expression patterns derived from microarray experiments. classifi circumvents two traditional limitations associated with microarray expression analysis – the effects of experimental and biological noise, and the variable depth of knowledge of individual genes in the probe set. a well-characterized gene has relatively precise annotations that reach deep into the go hierarchy, while a poorly-characterized gene is only annotated with high level terms. by capturing the entire ontology parentage of each gene, classifi overcomes limitations associated with variable depth-of-knowledge associated with most gene sets. in addition, the probability calculation used in classifi is relatively robust to the addition of experimental noise, allowing the use of less stringent data filtering approaches, which reduces the false negative discovery rate without the usual associated increase in false positives. indeed, increasing the sam fdr followed by classifi analysis increase the number of endocytosis-related genes while maintaining similar gene cluster classifications. our analysis focused on the aig-specific gene cluster # which was statistically classified as an intracellular transport-related gene cluster. inspection of the entire gene list leads to the hypothesis that gene cluster # classification reflects the broader biological process of bcr-mediated endocytosis, vesicle acidification and trafficking, and antigen processing and presentation. classifi should be considered a hypothesis-generating tool that requires experimental validation. several predictions of our hypothesis were confirmed experimentally, including ligand-specific endocytosis induction and mhc class ii expression. it should be noted that although we have used gene ontology annotation as a means of gene classification, any gene description scheme of interest could be used with the classifi approach to link experimental variables with legacy knowledge through gene expression microarray data. identification of putative components of bcr-mediated antigen capture, processing, and presentation based on the genes responsible for gene cluster # classification, we hypothesized that bcr engagement activates receptor-mediated endocytosis, vesicle acidification, vesicle trafficking, and antigen processing and presentation. we manually curated the aig-specific cluster gene list and found a total of genes, including those identified using classifi, that might be involved based on the molecular functions defined in other cell systems and biological processes from the literature. figure shows a schematic representation of the b cell antigen processing and presentation pathway indicating the putative roles played by these genes. several genes known to be involved in signal transduction were found in gene cluster #18; some have been demonstrated to play a role in bcr signaling while others are involved in signaling pathways in other cell types and are candidates for new bcr signaling components that stimulate the endocytic process. activation of protein kinases, including members of the map kinase family, in response to bcr engagement is well documented; thus strap, map4k and map2k may also play a role in bcr signaling. indeed, map4k is expressed in hematopoietic cells and is required for activation-induced t cell death following tcr stimulation, and map2k is required for il- production by macrophages and dendritic cells, and antigen-mediated activation of t cells. cd has been found to regulate the cellular response to bcr signaling either through the recruitment of the shp- phosphatase thereby dampening bcr signaling, or by cooperating with bcr signaling to prevent apoptosis in immature b cells. the dusp dual specificity phosphatase has been found to associate with the cd co-receptor and dampen tcr-mediated signaling in t cells by inactivation of map kinases, suggesting that it may regulate bcr signaling in a similar fashion. daam is involved in wnt signaling in xenopus embryogenesis. strap binds to the tgf beta type i receptor and inhibits downstream signaling. whether these proteins might play a role in regulating bcr signaling remains to be determined. the nf-atc isoform has been found to participate in the bcr-induced apoptosis in b-cell-derived burkitt's lymphoma cell lines, thus the presence of nfatc2ip and fkbp suggests that these interacting proteins may also be important in regulating ca++/calmodulin/calcineurin/nfat signaling in b cells. a large proportion of genes in gene cluster # encode proteins with roles in endocytosis, vesicle trafficking, vesicle acidification, molecular processing and protein secretion. in most cases these roles have been defined in other cell systems. since little is known about the molecular details of these processes in receptor-mediated antigen capture and mhc class ii-dependent antigen processing and presentation in b cells, the possibility that gene cluster # has produced a putative list of proteins that coordinate these activities is intriguing. snx a member of the sorting nexin family, has been found to interact with clathrin in skeletal muscle. clathrin has been implicated in facilitating membrane invagination during receptor-mediated endocytosis in b cells. the presence of snx in this cluster suggests that it may help mediate this process. the als protein acts as a guanine nucleotide exchange factor for rab and rac and stimulates early endosome fusion. rab proteins are small gtpases with homology to ras that play key roles in vesicle transport: rab has been found to localize near the apical and basolateral plasma membrane in epithelial cells of the kidney and intestine and rab22a has been found to localize to the plasma membrane and early endosomes. iqgap is a scaffolding protein with multiple protein-protein interaction domains and a gtpase activation protein domain that stimulates the hydrolytic activity of ras family members. iqgap has also been found to localize to areas of membrane ruffling and newly formed vesicles in astrocytoma cells. genes encoding homologs to several components of the vesicle-type proton pump were found in gene cluster # including atp6v0b, atp6v1g atp6v1c and atp6v1f and the proton pump accessory protein atp6ap additional proton pump components were also found to be induced in an aig-specific manner by rt-pcr. together with the vesicle-specific chloride channel clcn these proteins are likely to play a role in the acidification of the endocytic vesicles in preparation for their fusion with lysosomes to facilitate antigen hydrolysis. proton pump components can serve as a membrane anchor point for snare proteins. napa appears to mediate the tethering of these vesicles in preparation for fusion. rab also appears to play a role in vesicle fusion since mutations in rab lead to the accumulation of lipid-rich vesicles. several proteins involved in lysosome formation and antigen processing were found in gene cluster # dysbindin binds to dystobrevins and is a component of the biogenesis of lysosome-related organelles complex in mouse liver, which regulates trafficking to lysosomal organelles. mutations in the cln gene leads to a lysosomal storage disorder associated with the accumulation of lipopigment-laden vesicles. hexosaminidase a is a ph-sensitive hydrolase. a variety of cathepsins are involved in mhc class ii antigen processing and presentation. none of the probes for these genes passed our data filtering process, however the cathepsin inhibitor cystatin b was found in gene cluster # suggesting that it might play a role in regulating cleavage site selection by modifying the relative activities of proteases involved in antigen processing and peptide loading. hspbp may facilitate antigen processing by regulating the chaperone function of hsp which has been associated with the presentation of myelin basic protein though mhc class ii in multiple sclerosis. several genes involved in mhc class ii expression were found in the aig-specific gene cluster. atf is a transcription factor that activates the promoter of the class ii trans-activator. translocation of nascent mhc class ii peptide chains into the lumen of the endoplasmic reticulum involves the function of the sec translocation complex, the signal sequence receptor ssr and the signal peptidase spc calmegin is a ca++ binding chaperone protein with significant homology to calreticulin and calnexin, an er chaperone protein that regulates the assembly of mhc class ii with the ii invariant chain. vesicles containing peptide-loaded mhc class ii are transported to the plasma membrane for fusion and exposure on the cell surface, perhaps by traveling back through the tgn into the secretory pathway. the vsp protein has been found to facilitate this kind of retrograde tgn transport in yeast. in neurons, reticulon is associated with synaptophysin in tubulovesicular structures and may play a role in the process of vesicle secretion. cd an mhc class ii co-receptor, may play a role in regulating mhc receptor compartmentalization. genes found in gene cluster # encode proteins that have some connection with signaling, antigen capture, vesicle transport, vesicle acidification or mhc class ii expression, suggesting that activation of b cells through the bcr is inducing the expression of genes involved in mhc class ii antigen processing and presentation. the activation of class ii presentation by antigen receptor engagement in b cells has been described extensively. the findings reported here are novel in that microarray results combined with classifi analysis have provided a list of genes that may be involved in this important immunological process. finally, it is intriguing to note the presence of genes involved in the regulation of apoptosis and autoimmunity in gene cluster # given the known function of the bcr in negative selection and the induction of autoimmunity. transcriptional positive feedback the changes in mrna levels for these genes involved in vesicle trafficking might be necessary to stimulate this biological process in aig-stimulated b cells. however, the kinetics of the mrna changes compared with the rapid induction of endocytosis suggests that this may not be the case. elevated mrna levels for these genes was found at and hr, but not at or hr post-stimulation. however, bcr endocytosis was maximally stimulated by minutes. the rapid induction of endocytosis suggests that at least the initiation of this process is activated through post-transcriptional mechanisms. this finding suggests that the aig-specific transcriptional reprogramming observed in response to bcr stimulation may reflect positive feedback regulation in which expression of proteins that have already been activated is increased either as a means to amplify or sustain the process. perhaps components of this important biological process are present at low levels in resting cells, and levels increase in response to appropriate environmental cues. this kind of regulatory process might allow cells to respond rapidly to a broad set of variables in their changing environment while conserving energy and materials while in a resting state.  in this paper, we describe a bioinformatics analysis of a b cell microarray dataset using classifi to determine the biological significance of microarray gene clusters defined by ligand-specific b cell responses. importantly, results from laboratory experimentation support the hypotheses derived from this microarray data mining exercise. this work is a model for immunologists and other biologists for utilizing the growing field of bioinformatics for microarray data mining, hypothesis generation and hypothesis testing. furthermore, the results from this analysis provide a foundation for a more detailed understanding of the b cell antigen processing and presentation pathway. 
mhc class ii expression was found to be maintained specifically in response to bcr stimulation. classifi analysis revealed an overrepresentation of genes involved in ion and vesicle transport, including multiple components of the proton pump, in the bcr-specific gene cluster, suggesting that activation of antigen processing and presentation pathways is a major biological response to antigen receptor stimulation. furthermore, ligand-specific internalization of the bcr, a first step in b cell antigen processing and presentation, was demonstrated. furthermore, this analysis has identified at least thirty-eight candidate components of the b cell antigen processing and presentation pathway and sets the stage for future studies focused on a better understanding of the components involved in and unique to b cell antigen processing and presentation. these observations provide experimental validation of the computational approach implemented in classifi, demonstrating that classifi-based gene expression cluster analysis is an effective data mining tool to identify biological processes that correlate with the experimental conditional variables. in order to define the common and unique biological responses to ligand stimulation, we compared the gene expression changes induced in normal primary b cells by a panel of ligands using cdna microarrays and a statistical approach, classifi, which identifies significant co-clustering of genes with similar gene ontology™ annotation. proton pump components that were not included in the initial microarray data set were also upregulated in response to bcr stimulation in follow up experiments. activation of naïve b lymphocytes by extracellular ligands, e.g.
 activation of naïve b lymphocytes by extracellular ligands, e.g. antigen, lipopolysaccharide and cd ligand, induces a combination of common and ligand-specific phenotypic changes through complex signal transduction pathways. for example, although all three of these ligands induce proliferation, only stimulation through the b cell antigen receptor induces apoptosis in resting splenic b cells. in order to define the common and unique biological responses to ligand stimulation, we compared the gene expression changes induced in normal primary b cells by a panel of ligands using cdna microarrays and a statistical approach, classifi, which identifies significant co-clustering of genes with similar gene ontology™ annotation.  classifi analysis revealed an overrepresentation of genes involved in ion and vesicle transport, including multiple components of the proton pump, in the bcr-specific gene cluster, suggesting that activation of antigen processing and presentation pathways is a major biological response to antigen receptor stimulation. proton pump components that were not included in the initial microarray data set were also upregulated in response to bcr stimulation in follow up experiments. mhc class ii expression was found to be maintained specifically in response to bcr stimulation. furthermore, ligand-specific internalization of the bcr, a first step in b cell antigen processing and presentation, was demonstrated.  these observations provide experimental validation of the computational approach implemented in classifi, demonstrating that classifi-based gene expression cluster analysis is an effective data mining tool to identify biological processes that correlate with the experimental conditional variables. furthermore, this analysis has identified at least thirty-eight candidate components of the b cell antigen processing and presentation pathway and sets the stage for future studies focused on a better understanding of the components involved in and unique to b cell antigen processing and presentation. 