the adaptive immune response mainly depends upon b- and t - cells that originate from hematopoietic stem cells . b - cells mature in the bone marrow and are released into the peripheral blood to subsequently reach secondary lymphoid tissues . during the t - cell - dependent antibody response to exogenous antigens , b - cells undergo activation , forming the characteristic germinal center ( gc ) that is detectable in peripheral lymphoid tissues such as the spleen , tonsils , peyer 's patches , and lymph nodes [ 2 , 3 ] . gc formation requires the interaction between costimulatory b - cell - surface receptors with specific ligands expressed on t helper cells and/or antigen - presenting cells . antigen - activated b - cells undergo a progressive clonal expansion and differentiation into centroblasts in the dark zone of the gc where the process of somatic hypermutation leads to base - pair changes in the v(d)j region of rearranged genes that encode the ig - variable region of immunoglobulin chains . centroblasts then differentiate into centrocytes and migrate into the light zone of the gc where a strong process of selection eliminates by apoptosis the centrocytes presenting low - binding antibodies . selected centrocytes can then differentiate into plasma cells ( pcs ) , responsible for antibody production , or into memory cells . although the specific transcriptional program of nave b - cell expansion and differentiation into pcs is not yet fully understood , several transcription factors are functional differentiation markers . specifically , bcl6 , pax5 , blimp1 , nf-b , some stat family members , and ifn - induced factors are required for specific differentiation steps as demonstrated in knockout mice , where the deletion of some of these genes induced the lack of specific b - cell subsets [ 512 ] . recently , the ifn - induced factor ifi16 protein has been associated with b - cell dysfunction and autoimmune diseases suggesting a possible role for this protein in b - cell biology . ifi16 is a major member of the ifn - inducible pyhin protein family , which consists of human and mouse proteins that are characterized by their ability to induce interferon , nuclear localization , and hematopoietic expression . the pyhin proteins may , therefore , serve as receptors for microbial dna , providing new insights into infectious diseases . ifi16 contains two specific stretches of 200 amino acids ( hin-200 domain ) , designated a and b domains , at the c - terminus , and the pyd domain at the n - terminus [ 14 , 15 ] . the ifi16 gene encodes three protein isoforms that are generated from the translation of three separate mrnas , which are produced by alternative mrna splicing [ 1619 ] . in normal human bone marrow , ifi16 expression is detected in cd34 + hematopoietic stem cells and throughout differentiation into monocytes and lymphocytes ; however , ifi16 expression is downregulated when cd34 + hematopoietic stem cells differentiate into red cells , neutrophils , or eosinophils . several studies have demonstrated that ifi16 plays an important role in the modulation of cell proliferation , survival , and senescence . ifi16 negatively regulates the cell cycle through the binding and functional modulation of several molecules involved in cell cycle regulation such as p53 , rb , and p21 [ 15 , 1927 ] . in particular , ifi16 is associated with cell cycle arrest in g0/g1 and/or g2/m phases in some cell lineages [ 28 , 29 ] . ifi16 overexpression is also related to apoptosis activation [ 3032 ] , and the slow dividing hematopoietic progenitor cd34 + cells exhibit an approximately 4-fold increase in ifi16 expression with respect to the fast - dividing subset of the hematopoietic progenitor cd34 + cells . ifi16 expression is deregulated in autoimmune diseases and primary cancers [ 23 , 36 ] . although ifi16 expression can be regulated through treatment with many differentiation stimuli , ifi16 is primarily induced by interferon ( ifn ) types i and ii , and its expression is related to specific ifns and cell types . furthermore , ifi16 plays a direct role in ifn--mediated responses because it responds directly to intracellular exogenous dna in a toll - like receptor - independent fashion [ 39 , 40 ] . association with ifn activity indicates a possible involvement of ifi16 in some autoimmune diseases that feature high interferon levels . moreover , a possible relationship between ifi16 and prolonged nf-b activation that affects b - cell survival and induces b - cell cycle derangement especially through the p53 pathway has been suggested . however , there have been limited studies on ifi16 in lymphoma cell lines and multiple myeloma [ 4143 ] , and no data are available on normal b - cell subsets . in this study , we focused our attention on ifi16 expression in normal b - cell subsets . our goal was to assess the ifi16 expression patterns and their possible relationships with the most relevant transcription factors controlling b - cell development . whole blood samples were collected from healthy blood donors through venipuncture in edta - containing tubes after providing informed consent following the helsinki declaration . peripheral blood mononuclear cells ( pbmcs ) were separated using a ficoll gradient ( ficoll - hystopaque , pharmacia , uppsala , sweden ) . nave and memory b - cells were purified from healthy donor blood using a nave b - cell isolation kit ( stemcell , grenoble , france ) or a memory b - cell isolation kit ( miltenyi , auburn , ca , usa ) , respectively , following the manufacturers ' instructions . the nave and memory b - cells were analyzed using flow cytometry after the isolation procedure to determine the purity percentage of these b - cell subsets . cd19/cd27 and cd19/cd27 b - cells consisted of > 95% in purified memory and nave b - cells , respectively . we analyzed the gene expression profile ( gep ) data that were previously generated and reported from different subsets of human b - cells [ 44 , 45 ] . briefly , we analyzed the gep data from 25 samples of normal b - lymphocytes ( nave cells , n = 5 ; germinal center cells , n = 10 ; memory cells , n = 5 ; plasma cells , n = 5 ) . all data were obtained by using the affymetrix hg - u133 2.0 plus microarray ( affymetrix , inc . http://www.affymetrix.com/support/index.affx ) and are available at http://www.ncbi.nlm.nih.gov / projects / geo/. for further technical details , see . in particular , we focused on the expression of ifi16 , which was identified using three different probe sets with the hg - u133 2.0 plus genechip ( 206332_s_at ; 208966_x_at ; and 208965_s_at ) . the mean values from the three probes were used for the analyses . because the gep data were derived from different experiments , adequate normalization quality control was performed as previously reported and verified through box plot and ma plot consistency analyses . gep analyses were performed using genespring gx 12.0 software ( agilent technologies , santa clara , ca , usa ) [ 4446 ] . to assess the effects of cd40 signaling on ifi16 gene expression , we analyzed the previously reported gep data . briefly , these data were originally generated using retroviral transduction to induce cd40 signaling in burkitt lymphoma cell lines . the cel files that were originally available at geo dataset gse2350 were analyzed were analyzed using genespring gx 12.0 . supervised analysis was conducted as previously reported   using a p value and fold change cut - off of 0.05 and 2 , respectively , and a multiple test correction according to benjamini - hochberg was adopted . ifi16 interaction with master b - cell regulators ( selected based on their relevance for mature b - cell development according to the current literature , such as blimp1 , bcl6 , mta3 , pax5 , irf4 , irf8 , xbp1 , rela , relb , rel , oct - binding factor 1 ( pou2af1 ) , spib , bach2 , stat3 , stat5a , and stat5b ) was evaluated by calculating the coefficient of determination ( r ) and the pearson correlation ( see the statistical analyses section ) . only the genes showing a r > 0.5 and a pearson correlation significance with p value < 0.01 were selected for further analysis . the selected genes were then inferred by applying the aracne algorithm . to maximize the statistical significance , we referred to a large dataset of human normal and neoplastic b - cells as well as human b - cell lines that has been reported previously [ 45 , 48 ] and is available at geo datasets gse2350 and gse12195 aracne was performed using geworkbench software , with bootstrapping , at a p value threshold of < 0.01 before correction for multiple testing [ 45 , 4851 ] . in fact , based on our analysis , ifi16 expression was suppressed by other molecules in pcs , making them unsuitable for an appropriate evaluation of the relations between ifi16-bcl6 and ifi16-irf4 . for the nf-b pathway analysis we also studied the expression of well - known transcriptional targets , such as bcl2 , ccnd2 , ccr7 , cflar , il2 , irf4 , and nfkbia , which have been previously used to define nf-b activation by gep . in particular ( i ) we studied their expression levels ( normalized microarray data ) , ( ii ) we studied the mean value of their expression in each sample ( referred to as nf-b signature  ) , and finally ( iii ) we divided the analyzed samples into two groups ( nf-b+ and nf-b ) based on whether the expression levels of the nf-b signature were above or below the median value . ifi16 mrna expression analysis was performed through quantitative real - time rt - pcr ( qrt - pcr ) on rna extracted from peripheral blood purified b - cell subsets achieved from three healthy donors ( n = 3 , two men and one woman , age between 32 and 36 years ) . total rna was extracted from purified b - cell subsets using the high pure rna isolation kit ( roche , mannheim , germany ) and stored at 80c . then , total rna was reverse transcribed and amplified employing the quantitect sybr green rt - pcr kit ( qiagen , hilden , germany ) following the manufacturer 's procedure . in brief , sybr green real - time pcr assay was performed in a 20  l pcr mixture volume consisting of 10  l of 2x quantitect sybr green rt - pcr master mix ( qiagen ) , containing hotstartaq dna polymerase , 400  nm of each oligonucleotide primer , 0.2  l of 100x quantitect rt mix ( containing omniscript and sensiscript reverse transcriptases ) , and 100  ng of extracted rna . the ifi16 primer sequences   were as follows : ifi16f : 5-acaaacccgagaaacaatgacc-3 , nt 14641485 , ( rnarefseq nm_005531.2 ) and ifi16r : 5-gcatctgaggagtccgaaga-3 nt 15841565 , ( rnarefseq nm_005531.2 ) . the amplification was performed in a lightcycler ( roche , mannheim , germany ) with an rt step ( 1 cycle at 50c for 20  min ) followed by the initial activation of hotstartaq dna polymerase at 95c for 15  min and 40 cycles in three steps : 94c for 10  s ; 60c for 15  s ; and 72c for 30  s. the lightcycler 5.3.2 software determined the quantification cycle ( cq ) . the ifi16 amplicons were checked through a melting analysis , and a single sharp peak was detected ( tm = 82.5c ) . the relative ifi16 mrna expression levels and the normalization to ribosomal 18s rna were calculated using the ct method as described previously [ 34 , 35 , 54 ] . ifi16 protein expression was determined in nave and memory b - cells isolated from healthy donor peripheral blood through flow cytometry and western blot procedures . for flow cytometry , purified b - cell subsets were achieved from five healthy donors ( n = 5 , three men and two women , age between 30 and 36 years ) . purified nave or memory b - cells were fixed in 1% pf for 30 minutes at room temperature . after several washes in pbs , the cells were treated for 45 minutes with 0.2% saponin ( sigma , st . louis , mo , usa)/pbs and then with a rabbit anti - ifi16 antibody ( sigma ) or , as a negative control , with a rabbit anti - hiv-1 p24 antibody ( diatheva , pesaro , italy ) diluted 1  :  40 in 0.2% saponin / pbs for 30 minutes at room temperature . after washing with pbs , the samples were stained with fitc - anti - rabbit igg ( dako , copenhagen , denmark ) diluted 1  :  100 in 0.2% saponin / pbs for 30 minutes at room temperature . the samples were analyzed using facscalibur flow cytometry ( becton - dickinson ) and cellquest software ( becton - dickinson ) . western blot analysis was performed on purified nave or memory b - cells achieved from healthy donors ( n = 3 ; two men and one woman , age between 32 and 36 years ) . the cells were lysed in laemmli buffer and equivalent amounts of proteins per sample were subjected to electrophoresis on a gradient 412% sodium dodecyl sulfate- ( sds- ) acrylamide precast gel ( thermo scientific , waltham , ma , usa ) . the gel was then blotted onto a nitrocellulose membrane , and equal loading of protein in each lane was checked by staining of the blot membrane with 0.1% ponceau s followed by destaining with distilled water . ifi16 protein detection was performed using rabbit anti - ifi16 antibody ( sigma , st . louis , mo , usa ) at 1  :  200 dilution , in blocking buffer ( 3% bsa in tbs/0.05% tween 20 ) for 2 hours and after several washes in tbs/0.05% tween 20 , the nitrocellulose membrane was treated with alkaline phosphatase - conjugated goat anti - rabbit igg ( sigma ) at 1  :  1000 dilution in blocking buffer for 2 hours . immunoreactive bands were visualized with bcip / nbt solution ( sigma ) . as control , tubulin protein was detected by mouse anti - tubulin monoclonal antibody ( mab ; sigma ) at 1  :  200 dilution and revealed by goat anti - mouse igg ( sigma ) at 1  :  1000 . double immunofluorescence labeling on processed paraffin sections of reactive lymph nodes was performed as described previously . pressure cooked , microwave - treated slides were incubated for 1 hour with the following mixtures of antibodies directed to ( i ) cd20 ( mouse mab , clone l26 , dilution 1  :  200 , dako ) and ifi16 ( rabbit antibody , dilution 1  :  30 ; sigma ) ; ( ii ) bcl6 ( mouse mab , clone pg - b6p , dilution 1  :  30 , m7211 , dako ) and ifi16 ; ( iii ) prdm1/blimp1 ( mouse mab , clone rosi95 g , dilution 1  :  10 , kindly provided by dr . roncador , cnio , madrid ) and ifi16 ; and ( iv ) cd138 ( rabbit antibody , dilution 1  :  400 , rb 9422-p , neomarkers ) and anti - prdm1/blimp1 . the slides were then incubated in the dark for 1 hour with the appropriate fluorophore - conjugated secondary antibodies ( alexa fluor 568-conjugated goat anti - mouse igg2a , invitrogen , carlsbad , ca , usa , for cd20 , and alexa fluor 488-conjugated goat anti - rabbit igg , invitrogen , for ifi16 and cd138 , and alexa fluor 568 , goat anti - mouse igg1 , invitrogen for bcl6 and prdm1/blimp1 ) . the micrographs were obtained using an olympus bx61 microscope equipped with an olympus dp-70 digital camera ; image acquisition , evaluation , and color balance were performed using cellf software . statistical analyses were performed with the statview 5.0 software package ( sas institute inc , cary , nc ) and wessa , pearson correlation ( v1.0.3 ) in free statistics software ( v1.1.23-r6 ) , office for research development and education , url http://www.wessa.net / rwasp_correlation.wasp/. anova , unpaired t - tests , and , when appropriate ( specifically , when the sample size was less than 10 cases in at least 1 group ) , a nonparametric ( mann - whitney ) test were adopted for gep data analyses for the comparison of ifi16 expression in different subgroups . the limit of significance for all analyses was defined as p < 0.05 ; two - sided tests were used in all calculations . the possible relationship between the expression of ifi16 and other genes ( transcription factors regulating b - cell development ) relations were regarded as significant for r > 0.50 and pearson correlation with a p value < 0.01 . to investigate whether ifi16 was modulated during b - cell functional differentiation , we studied ifi16 gene and protein expression in normal b - cell subsets . ifi16 gene expression was analysed using data previously generated by dna - microarrays [ 44 , 45 ] . nave and memory b - cells displayed a significantly higher amount of ifi16 mrna than gc b - cells ( nave versus gc p = 0.0062 ; memory versus gc p = 0.0131 ) or plasma cells ( p < 0.0001 ; figure 1 and table 1 ) . conversely , no significant differences in ifi16 mrna expression between nave and memory b - cells were detected ( p = 0.78 ) , although a consistent variation between individuals was observed in memory b - cells ( figure 1 ) . finally , gc b - cells exhibited significantly higher ifi16 mrna levels than pcs ( p < 0.0001 ; figure 1 and table 1 ) . as ifi16 can also be regulated posttranscriptionally , its expression , at the protein level , was analyzed in lymph nodes using an indirect immunofluorescence assay . double staining with antibodies directed against ifi16 and the b - cell marker cd20 clearly demonstrated ifi16 protein in the lymph node compartments . as expected , there was strong cd20 staining in the germinal center and in the mantle zone , and ifi16 was detectable inside the nucleus of cd20 positive b - cells ( figures 2(a)2(c ) ) . in accordance with the gene expression results , ifi16 staining was clearly brighter in the mantle zones , which are normally populated by nave and memory cells , than in the gcs , which are basically composed of centroblasts and centrocytes ( figure 2(c ) ) . because the mantle zones can be quite heterogeneous in their composition , we also evaluated ifi16 expression in nave and memory b - cell subsets purified from healthy donor peripheral blood samples using immunomagnetic procedures . using flow cytometry , slightly higher ifi16 expression levels in memory cells compared with nave b - cells western blot analysis of protein extracts from peripheral blood nave and memory b - cell subsets demonstrated the presence of the three ifi16 isoforms generated by alternative splicing ( figure 3 ) . consistently , qpcr assays demonstrated that the ifi16 mrna content was similar in peripheral blood nave and memory b - cell subsets ( figure 3 ) , thus confirming the lymph node observations . taken together , these results indicated that ifi16 expression was modulated throughout b - cell differentiation with a progressive downregulation during the gc reaction and differentiation to pcs . the observation of different expression levels of ifi16 in b - cell subsets and , in particular , the significant ifi16 mrna decrease in the crucial gc b - cell subset prompted us to analyse whether ifi16 is regulated by major transcription factors involved in b - cell regulation and differentiation . we considered the transcription factors that are more strictly involved in the modulation of gc cells , including blimp1 , bcl6 , mta3 , pax5 , irf4 , irf8 , xbp1 , nf-b , pou2af1 , ets family members ( as spib ) , bach2 , stat3 , stat5a , and stat5b ( for details on their expression levels in b - lymphoid subpopulations , see figure 4 ) . first , we calculated the linear regression coefficient of correlation ( coefficient of determination , r ) between the expression levels of ifi16 versus the different transcription factors and selected only those transcription factors with r > 0.5 . second , we calculated the pearson correlation and filtered only those with a significant correlation and a p value < 0.01 . we found that ifi16 mrna levels inversely correlated with xbp1 , pou2af1 , blimp1 , and bcl6 expression , whereas irf4 , stat3 , rel , spib , rela , relb , stat5b , and stat5a showed a direct correlation with ifi16 expression . mta3 , pax5 , irf8 , and bach2 did not show any significant correlation ( table 2 ; figure 5 ) . to validate this observation , we performed double immunostaining assays for ifi16/bcl6 or ifi16/blimp1 in a series of reactive lymphoid tissues . of note , immunostaining confirmed the inverse relationship between ifi16 and blimp1 and between ifi16 and bcl6 ( figure 6 ) . to assess whether the identified transcription factors could directly target the ifi16 gene , we used a recently developed bioinformatic algorithm ( aracne ) , suitable to identify transcriptional targets [ 9 , 49 ] . interestingly , aracne indicated ifi16 as a possible direct target of bcl6 , relb , stat5b , and pou2af1 but not of blimp1 , xbp1 , irf4 , stat3 , rel , spib , rela , and stat5a ( table 2 ; figure 7 ) . notably , these results were consistent with those reported by basso and coworkers , who studied the bcl6 transcriptional network . altogether , these data indicated that ifi16 modulation during mature b - cell differentiation was strictly associated with the function of different transcription factors . particularly , ifi16 downregulation during the germinal center ( gc ) transition appeared to be due to bcl6-mediated transcriptional repression . in contrast , in both nave and memory b - cells following b - cell receptor ( bcr ) and cd40 stimulation , nf-b and stats protein activation were related to ifi16 levels along with bcl6 downregulation . finally , in plasma cells ( pcs ) , the combined effects of blimp1 , pou2af1 , and xbp1 are likely to maintain the lowest levels of ifi16 gene expression . the nf-b pathway is involved in b - cell biology and differentiation and some reports have shown that ifi16 can upregulate nf-b activity in endothelial cells [ 58 , 59 ] . therefore , based on the evidence of a correlation between ifi16 and rela , relb , and rel , we further analysed this possible interaction in b - cell subsets . because the functionality of nf-b subunits ( rela , relb , and rel ) is quite complex and can not simply be evaluated by detecting changes in their gene expression , we also analyzed the activity of the nf-b pathway by studying the expression of nf-b target genes , as reported previously . higher ifi16 levels were found in the presence of nf-b activation ( p < 0.0001 ; table 3 ; figure 8) , confirming the correlation suggested by the transcriptional levels of the three rel family molecules . as the relationship between ifi16 and nf-b is not yet clear , except in endothelial cells , we studied the effects of cd40 ( a well - known nf-b activator in human b - cells ) signalling induction on ifi16 levels to determine whether ifi16 is an upstream activator or a downstream target of nf-b . to do so , we analyzed gep data that were originally generated in burkitt lymphoma ( bl ) cell lines in which cd40 signalling was induced by viral transduction to evaluate the early events after cd40 stimulation   ( see supplementary table 1 in supplementary material available online at http://dx.doi.org/10.1155/2015/747645 ) . although we observed clear activation of nf-b with increased expression of different nf-b molecules ( including nfkb2 and nfkbia ) and nf-b transcriptional targets ( including icam1 , traf1 , cflar , irf4 , and tnfaip3 ) [ 6164 ] , we did not find significant modulation of the ifi16 gene ( p = 0.8 ; fold change , 1.04 ) . together , these results indicate a significant association between nf-b activity and ifi16 expression but apparently excluded a direct effect of nf-b on ifi16 expression . therefore , our data support the concept that ifi16 can regulate nf-b in human b - cells , as already demonstrated in endothelial cells [ 58 , 59 ] . in this paper , we have analyzed ifi16 mrna and protein expression levels in normal b - cell subsets and investigated the correlations between levels of ifi16 and the expression of transcription factors known to play a relevant role in b - cell physiology and differentiation . specifically , we used gep analysis in b - cell subsets purified from lymph nodes to explore the transition from nave to either memory or pcs throughout the gc reaction . overall , gep analysis showed that changes in ifi16 expression were related to cell differentiation stages . the passage from nave b - cell subsets to proliferating gc cells was associated with a significant downregulation of ifi16 expression . ifi16 mrna levels also declined dramatically when gc cells differentiated into pcs , whereas the shift from gc cells to memory cells demonstrated an increase in ifi16 expression that was comparable to those observed in nave b - cell subsets . because ifi16 can also be regulated posttranscriptionally , we evaluated ifi16 protein expression and confirmed the results of the gep analysis . immunofluorescence staining of ifi16 in lymph nodes showed a clear decrease in fluorescence in germinal center cd20 b - cells with respect to cd20 b - cells in the mantle zones . these observations indicate that ifi16 expression is regulated in gc cells and its modulation correlated with b - cell differentiation fate . to gain further insights into the control of ifi16 expression in gc cells , we compared ifi16 expression with the expression of pivotal transcription factors involved in gc biology and differentiation . gep analysis showed that ifi16 expression was significantly related to the expression of the transcription factor bcl6 . this observation was confirmed through bioinformatic inference of the ifi16 regulatory network using aracne , which indicated a direct relationship between bcl6 and ifi16 . moreover , double immunofluorescence analysis indicated low ifi16 protein expression when high bcl6 levels were detected in b - cell compartments . consistently , a recent study indicated that ifi16 belongs to the first neighborhood of bcl6 , within its transcriptional network . bcl6 is a nuclear phosphoprotein that is specifically expressed in the gc in the b - cell lineage and is detectable in the centroblasts and in the majority of centrocytes . bcl6 is a master regulator of gc constitution , and bcl6-deficient mice have normal b - cell development but no gc formation [ 5 , 7 ] . the deregulated expression of bcl6 induced an increase in gc formation in transgenic mice . in gc , bcl6 regulates differentiation , apoptosis , genotoxic stress response , and cell cycle progression [ 48 , 6570 ] . specifically , bcl6 upregulates proliferation of gcs by suppressing the expression and/or the activation of genes involved in negative cell - cycle regulation such as tp53 , pias2 , and p21 . in addition , bcl6 supports the dna breaks induced by shm and csr , suppressing the sensing of dna damage by atm and rad3 inhibition [ 4 , 65 ] . conversely , ifi16 downregulates cell proliferation and reinforces genotoxic stress responses by binding several molecular partners including p53 [ 20 , 29 , 7274 ] . therefore , it is conceivable that ifi16 transcription inhibition , which is directly controlled by bcl6 during the gc transition , may be relevant to the correct control of cell cycle progression and dna damage responses in gc cells . in support of this hypothesis , ifi16 acts as a dna sensor that activates genes involved in cell cycle inhibition and dna repair [ 29 , 75 ] . moreover , ifi16 might act as a scaffold protein that is generally associated with the suppression of cell proliferation by causing cell death or by triggering senescence [ 21 , 23 , 26 , 27 , 32 , 76 ] . we showed evidence that , during gc transition , pou2af1 could negatively regulated ifi16 as well . on the other hand , no correlation was observed with other transcription factors that play a major role in gc formation [ 10 , 7779 ] , such as bach2 and irf8 . conversely , spib , stat3 , and stat5a showed a positive correlation , supporting the hypothesis that they may contribute to ifi16 expression in nave and memory cells ( figure 9 ) . interestingly , a relationship between stat3 activation and ifi16 expression during cell apoptosis was detected in medullary thyroid carcinoma and mammary epithelial cells . this finding indicates the possible existence of a more general regulatory network between stat3 and ifi16 [ 80 , 81 ] . notably , by using aracne , a robust bioinformatic algorithm [ 48 , 49 , 51 ] , we have defined a possible type of relationship between ifi16 and other molecules . in particular , we have shown evidence of a direct interaction between ifi16 and either bcl6 , stat5b , pou2af1 , or relb and an indirect relationship ( i.e. , mediated by intermediate factors ) for stat3 , xbp1 , rel , spib , blimp1 , rela , and stat5a . interestingly , ifi16 gene expression after the gc transition was regulated differently in pcs and memory b - cells . in fact , ifi16 was strongly downregulated in pcs and upregulated in memory b - cells . these two b - cell subsets originate from gc centrocytes cells upon bcr - driven signaling activation . although bcl6 is shut off in both cases , the terminal differentiation into plasma cells also requires pax5 inactivation and the induction of the transcriptional repressor blimp1 [ 4 , 82 ] . blimp1 has been recognized as a pivotal regulator of the transition between gc and plasma cells . in fact , mice with a b - cell - specific deletion of prdm1 , which is the gene encoding blimp1 , do not generate pcs , and transient transfection experiments with blimp1 expression vectors can determine plasmablast differentiation . by testing the possible connection between ifi16 and blimp1 genes accordingly , we documented their mutually exclusive expression through double - staining immunofluorescence assays . however , aracne analysis failed to identify a direct link between ifi16 and blimp1 , suggesting that this relationship is not direct but is likely mediated by an additional factor . in pcs , in particular , pou2af1 showed a very strong correlation with ifi16 and was indicated by aracne analysis as directly interacting with ifi16 . in parallel , the analysis of other factors such as mta-3 and pax5 that are involved in the regulation of gc differentiation to pcs or memory b - cell subsets did not show any significant association . these results may suggest that ifi16 mrna levels are sustained by spib and stats proteins , whereas bcl6 is inhibited , and xbp1 , pou2af1 , and blimp1 are absent . in contrast , ifi16 expression is negatively regulated during the transition to gc and during differentiation to pcs by means of a few major negative regulators such as bcl6 , xbp1 , pou2af1 , and blimp1 . this particular regulation of ifi16 expression in pcs may have some interesting parallels with myeloid progenitor - derived cell lineages . ifi16 is expressed in cd34 + hematopoietic progenitors and disappears when the cells differentiate to megakaryocytes and erythrocytes [ 14 , 17 ] . these observations indicate that ifi16 is tightly regulated in blood cell lineages and plays a regulatory role during the early step of progenitor cell differentiation ; however , ifi16 is lost in some lineages , especially when these cells reach their terminal differentiation . nf-b is a transcription factor that is involved in cell survival and activation of b - lymphocytes [ 47 , 84 ] . its activation is particularly evident upon b - cell activation , which results in the translocation of active nf-b into the nucleus where it regulates target genes . nf-b activation is particularly detectable after b - cell activation , which results in the translocation of active nf-b into the nucleus where it regulates target genes . using gep analysis , we observed that significantly higher ifi16 gene expression levels accompanied higher nf-b signature . in gcs and pcs , both ifi16 mrna levels and nf-b signaling decreased , whereas ifi16 mrna levels and nf-b signaling were restored in nave and memory cells . in the latter two cell subsets , nf-b activation is sustained , at least in part , by bcr and cd40 signaling . therefore , we studied the effects of the cd40 downstream cascade on ifi16 mrna levels in human b - cells to assess whether nf-b activation could directly induce changes in ifi16 expression . despite clear evidence of nf-b induction as a result of cd40 stimulation , importantly , consistent with our aim , the experiment was originally designed to detect the early events of cd40 stimulation . therefore , indirect effects ( such as those depending on the subsequent activation of irf4 and consequent bcl6 downregulation ) were not observed . overall , these results indicated that ifi16 might modulate nf-b in b - lymphocytes but not vice versa . nonetheless , as our results are based on gene expression modulation , we can not exclude a more complex functional interaction at the protein level . these data are in line with previous studies demonstrating that ifi16 positively regulates nf-b activation in endothelial cells [ 58 , 59 , 64 , 8587 ] . ifi16 over expression , indeed , in huvec endothelial cell model , triggers the nf-b complex activation through the inhibition of ib transcription and expression . in addition , the study of ifi16 activity as dna sensor indicated that viral and bacterial dnas activated ifi16 via hin domains , independently by tlr regulation [ 39 , 75 ] . ifi16 sensed the presence of viral or bacterial dna and restricted viral replication of several viruses including hsv , hcmv , ebv , and hiv [ 40 , 88 , 89 ] . in this experimental context [ 40 , 75 ] , it has been determined that ifi16 interacts with sting to modulate positively nf-b and , then , induce innate response genes [ 40 , 75 , 88 ] suggesting a complex relationship between ifi16 and nf-b . this paper is the first to describe the ifi16 expression pattern in normal human b - cells at the mrna and protein levels . although further studies are required to investigate the specific functions of ifi16 in b - cells , the detection of significant changes in ifi16 expression during differentiation stages along with its interaction with several transcription factors , including nf-b and bcl6 , involved in the b - cell biology , suggest an important role of ifi16 in this cell model .
the interferon - inducible dna sensor ifi16 is involved in the modulation of cellular survival , proliferation , and differentiation . in the hematopoietic system 
 , ifi16 is consistently expressed in the cd34 + stem cells and in peripheral blood lymphocytes ; however , little is known regarding its regulation during maturation of b- and t - cells . 
 we explored the role of ifi16 in normal b - cell subsets by analysing its expression and relationship with the major transcription factors involved in germinal center ( gc ) development and plasma - cell ( pc ) maturation . 
 ifi16 mrna was differentially expressed in b - cell subsets with significant decrease in ifi16 mrna in gc and pcs with respect to nave and memory subsets . 
 ifi16 mrna expression is inversely correlated with a few master regulators of b - cell differentiation such as bcl6 , xbp1 , pou2af1 , and blimp1 . 
 in contrast , ifi16 expression positively correlated with stat3 , rel , spib , rela , relb , irf4 , stat5b , and stat5a . 
 aracne algorithm indicated a direct regulation of ifi16 by bcl6 , stat5b , and relb , whereas the relationship between ifi16 and the other factors is modulated by intermediate factors . 
 in addition , analysis of the cd40 signaling pathway showed that ifi16 gene expression directly correlated with nf-b activation , indicating that ifi16 could be considered an upstream modulator of nf-b in human b - cells .