BACKGROUND
t lymphocyte activation requires recognition through the antigen-specific t cell receptor /cd <dig> complex of foreign peptides presented by self-mhc molecules on antigen presenting cells in context with co-stimulatory signals mediated by co-stimulatory receptors such as cd <dig>  <cit> .
the concurrent ligation of these receptors initiates signal transduction pathways that lead to the induction of a complex array of kinases and phosphatases, and the downstream activation of transcription factors such as nf-κb, ap- <dig> and nfat which play a critical role in reprogramming gene expression  <cit> .
the overall result is the proliferation, differentiation and activation of t cells.
however, t cells stimulated via the tcr/cd <dig> complex alone do not become fully activated and can become anergic or even apoptotic  <cit> .
transcriptional changes during t cell stimulation are tightly regulated by a variety of mechanisms involving not only interactions between a complex network of transcription factors and cis-regulatory dna regions, but also epigenetic changes in chromatin structure including acetylation, phosphorylation, methylation and ubiquitylation  <cit> .
accumulating evidence suggests an important role for the enzyme poly polymerase- <dig>  in the regulation of gene expression during development and in response to specific cellular signals  <cit>  working at different levels.
parp- <dig> belongs to a family of enzymes  that, using nad+ as a substrate, synthesize and transfer homopolymers of adp-ribose onto glutamic acid residues of acceptor proteins mainly involved in chromatin structure and dna metabolism.
polyation is terminated by the release of extensively polyated  parp molecules from dna.
adp-ribose polymers are then subjected to degradation by poly glycohydrolase   <cit> .
polyation is therefore an immediate, covalent, but transient post-translational modification of cellular proteins playing and important role in epigenetic regulation of chromatin structure and gene expression under physiological conditions in which the integrity of the dna is maintained  <cit> .
parp- <dig> activity, responsible for almost 90% of polylation in the cell, might modulate gene expression through polyation of its partner proteins or by its physical association with relevant proteins such as transcription factors.
regulation by parp- <dig> has been described for different transcription factors such as nf-κb  <cit> , e2f- <dig>  <cit> , c/ebpalpha  <cit> , yy- <dig>  <cit> , rna polymerase ii-associated factors  <cit> , p <dig>  <cit>  and nfat  <cit> .
indeed, parp- <dig> and polyation play a critical role in the expression control of multiple nf-κb dependent genes involved in the inflammatory response  <cit> .
in addition, different studies have analysed the effects of parp- <dig> deficiency on gene expression in fibroblasts  <cit> , cardiomyocytes  <cit> , leukemia cells  <cit> , glia  <cit> , endothelial cells  <cit> , embryonic stem cell lines and liver tissue  <cit>  at the genome-wide level.
recently, we have demonstrated that parp- <dig> is activated during t cell activation, modulating the activity of the nfat transcription factor  <cit> .
in the present study we use oligonucleotide microarray analysis to gain more insight into the role played by parp- <dig> during the gene expression reprogramming that takes place in t cells upon activation with anti-cd3-stimulation alone, or in combination with anti-cd <dig> co-stimulation.
these two stimulation conditions would allow us to analyse the role of parp- <dig> in the modulation of genes upon nfat activation under conditions in which both ap- <dig> and nf-κb are fully activated  or poorly activated .
t cells co-stimulated with anti-cd <dig> + anti-cd <dig> develop an overlapping transcription network with anti-cd <dig> stimulated t lymphocytes, but co-stimulatory signalling quantitatively alters components of the tcr/cd <dig> complex-induced signals  <cit> .
therefore, parp- <dig> could synergize with different transcription factors in order to stimulate or repress gene expression in both stimulation conditions.
we have identified several groups of genes, whose expression is modulated by parp- <dig> either in a positive or a negative manner.
interestingly, parp- <dig> deficiency significantly alters expression of genes associated with the immune response such as chemokines and genes involved in the th1/th <dig> balance.
RESULTS
transcriptional programs in parp-1-/- vs parp-1+/+ t cells in response to antigen receptor stimulation
in order to study the role of parp- <dig> in the transcriptional regulation of t cells, we have analysed the gene expression profiles in splenic t cells derived from parp-1-wild-type  and parp-1-deficient  mice under both resting and antigen receptor stimulation conditions, using oligonucleotide microarray technology.
total rna prepared from resting or stimulated cells was purified, reverse transcribed and amplified by in vitro transcription in the presence of biotinylated nucleotides, and the labeled crna was hybridized on affymetrix oligonucleotide chips  containing over  <dig> probe sets representing transcripts from over  <dig> well-characterized mouse genes.
only those differences in rna abundance that were reproducible in independent experiments with different batches of cells and represented by a change of  <dig> -fold or greater  were considered.
at a basal level, we found  <dig> genes  that differed in their expression between parp-1+/+ and parp-1-/- t cells.
this percentage is quite similar to the change in the basal expression level observed between primary parp-1+/+ and parp-1-/- fibroblasts  <cit> .
the expression of  <dig> of these genes in t cells was higher in parp-1-/- than in parp-1+/+ t cells , while the expression of  <dig> genes, obviously including the parp- <dig> gene itself, was lower in parp-1-/- compared to parp-1+/+ t cells  .
the specific genes that were differentially expressed in parp-1-/- vs parp-1+/+ t cells are listed in the additional files  <dig> and  <dig>  in addition, all data are deposited in the arrayexpress database  <cit>  with accession number e-mexp- <dig>  we did not observe genes overlapping between the report on primary fibroblast and our present results.
similar absence of overlap has been reported recently by ogino et al, between fibroblasts and es cells  <cit> .
this may be possibly due to differences in the cell linages examined in these different experiments.
a genes were classified as parp- <dig> dependent genes when their expression was change by a factor of at least  <dig>   between parp-1+/+ and parp-1-/- cells in the microarrays analysis.
bpositive regulation was considered when the expression of a particular gene was higher in the presence of parp- <dig> than in its absence.
likewise, genes expressed at higher level in the absence of parp- <dig> than in its presence were considered negatively regulated by parp- <dig>
to characterize the gene expression program in t cells responding to antigen receptor stimulation, t cells from both genotypes were subjected to stimulation for  <dig>  h with a plate-bound antibody to cd <dig> alone, or in combination with an antibody to cd <dig> and expression results were normalized to the unstimulated conditions.
short stimulation times have been shown to induce important changes in t cells gene expression programs  <cit> .
cd <dig> stimulation modulated a significant number of early-response genes   in parp-1+/+ t cells, up-regulating  <dig> and concomitantly down-regulating  <dig> transcripts, while in parp-1-/- t cells  <dig> of genes  were modulated, up-regulating  <dig> genes and down-regulating  <dig> transcripts.
on the other hand, cd <dig> stimulation in the context of cd <dig> co-stimulatory signal, modulated a significant number of early-response genes   in parp-1+/+ t cells, up-regulating  <dig> and concomitantly down-regulating  <dig> transcripts, while in parp-1-/- t cells  <dig> of genes  were modulated, up-regulating  <dig> genes and down-regulating  <dig> transcripts.
identification of parp-1-dependent genes during early t cell activation
transcriptional changes during t lymphocyte activation have also been characterized in the past  <cit> , and it is beyond the scope of this paper to provide a discussion of all of the observed changes and their biological implications.
however, comparison of the gene expression programs evoked by activation in parp-1+/+ and parp-1-/-t cells shows that  <dig> early-response genes to anti-cd <dig> seem to be parp-1-dependent  and regulated by parp- <dig> either in a positive   or in a negative manner   .
likewise, in the presence of co-stimulation , the expression of  <dig> genes is also regulated by parp- <dig> either in a positive  or in a negative manner  .
in addition, these data are listed in the additional files  <dig> and  <dig>  interestingly, response to anti-cd <dig> alone resulted in a higher number of genes negatively regulated by parp- <dig> than those regulated positively.
however, anti-cd <dig> stimulation in the presence of anti-cd <dig> co-stimulation produces the opposite effect, favouring expression of genes regulated positively by parp- <dig> .
these results could be related with a different role of parp- <dig> in the regulation of specific transcription factors involved in those signalling pathways.
thus, t cell stimulation via the tcr/cd <dig> complex alone seem to be sufficient for nfat but not for optimal nf-κb or ap- <dig> activation.
we have recently demonstrated that parp- <dig> inhibition seems to modulate in an opposite manner nfat and nf-κb-dependent transcriptional activity in t cells, improving nfat  <cit>  and inhibiting nf-κb activities .
however, the complexity of the nfat and nf-κb families, together with the regulation of genes by both transcriptional factor members precludes ascribing the described parp-1-dependent genes to a unique and direct effect on nfat or nf-κb dependent transcriptional activity.
twenty one genes were dependent on parp- <dig> for response to both anti-cd <dig> and anti-cd <dig> + anti-cd <dig> stimulation .
eleven of these genes were positively regulated by parp- <dig> while  <dig> of them were negatively regulated by parp- <dig> in response to either anti-cd <dig> or anti-cd <dig> + anti-cd <dig> stimulation.
however,  <dig> genes  were regulated positively by parp- <dig> in response to cd <dig> stimulation alone, but the same genes were regulated negatively by parp- <dig> in the presence of co-stimulation through cd <dig>  likewise,  <dig> genes  were regulated negatively by parp- <dig> in response to cd <dig> stimulation but positively by parp- <dig> in response to anti-cd <dig> + anti-cd <dig> stimulation .
we confirmed expression changes for some genes by real-time pcr.
although there were differences in the absolute fold-change values detected by the two methods, pcr results correlated well with the differential gene expression data produced using affymetrix genechips .
automatic functional annotation of parp-1-dependent genes in response to either anti-cd <dig> or anti-cd <dig> + anti-cd <dig> stimulation was performed based on ingenuity software.
although this type of annotation is very useful to obtain a description of the transcriptome, it can be misleading if the absolute number of genes that fall into each class is used as the only factor for selection of relevant classes.
consequently, we identified which gene-ontology  terms were significantly over-represented in each activation condition .
as shown in the examples in figure  <dig>  more parp-1-dependent genes than expected by chance were involved in certain processes or functions.
the bars indicate whether there are more parp-1-dependent genes with a given go term than expected with a random distribution in each activation condition.
interestingly, the highest differences were found for genes involved in lymphocyte activation and immune response, with little differences in genes involved in metabolism.
parp- <dig> deficiency affects the expression of genes involved in the immune response pathway in t cells
a subset of parp- <dig> dependent genes in response to anti-cd <dig> stimulation  and in response to anti-cd <dig> + anti-cd <dig> stimulation  encode for molecules involved directly in the immune response pathway  as determined using the ingenuity software.
to validate the array data, we also evaluated expression of several of these genes by quantitative real-time pcr.
although there were differences in the absolute fold-change values detected by the two methods, pcr results correlated well with the differential gene expression data produced using affymetrix genechips .
discordant results were found between the arrays and pcr for only gbp <dig> gene in response to anti-cd <dig> + anti-cd <dig> stimulation.
in response to stimulation through cd <dig> alone or in combination with mab to cd <dig> a significantly increased expression of il- <dig> was detected in parp-1+/+ compared to parp-1-/- t cells .
this result was further confirmed by real-time pcr .
in addition, t cells purified from parp-1+/+ and parp-1-/- mice were stimulated for  <dig> hours with anti-cd <dig>  alone or in combination with anti-cd <dig> mab, and il- <dig> released in the supernatants was measured by elisa .
the results verified the effect of parp- <dig> deficiency on the expression levels of il- <dig> obtained at the mrna level in the microarray and real-time pcr experiments.
overall, our data suggest an important role for parp- <dig> in the regulation of il- <dig> expression.
il- <dig> is a major th <dig> effector cytokine and a key promoter of th <dig> development  <cit> .
th <dig> and th <dig> cells are characterized by the types of cytokines that they produced.
ifn-gamma is the hallmark of th <dig> cells which are involved in the immune response against intracellular microoganisms.
on the other hand, th <dig> effector cells secrete il- <dig>  il- <dig> and il- <dig> being involved in immune response to helminth infections as well as to ige-dependent hypersensitivity reactions.
interestingly, the expression of ifn-gamma mrna seem to be increased in parp-1-/- t cells.
expression of several th <dig> chemokines such as xcl <dig>  ccl <dig> and ccl <dig> seem to be also increased en parp-1-/- t cells in response to anti-cd <dig> + anti-cd <dig> activation .
therefore, the absence of parp- <dig> seems to bias t cell response to a th <dig> phenotype.
however, our microarray data did not reveal any difference in the expression of other th <dig> cytokines such as il- <dig>  il- <dig> and il- <dig> between parp-1-/- and parp-1+/+ t cells, suggesting that parp- <dig> could specifically control il- <dig> gene expression which might act on th <dig> differentiation by supressing il-12br expression and ifn-gamma secretion.
previous studies have demonstrated the requirement of slam in il- <dig> production by t cells  <cit> .
similarly, irf <dig> gene expression was also found to be higher in parp-1+/+ than in parp-1-/- t cells after stimulation with anti-cd <dig> + anti-cd <dig> .
irf <dig> seems to enhance il- <dig>  but not il- <dig> promoter activity  <cit> .
whether parp- <dig> controls il- <dig> expression through an interaction with cis-regulatory elements or chromatin remodelling at il- <dig> locus remains to be elucidated.
reduction of il- <dig> production by lung cells has been reported in ova-challenged parp-1-/- mice compared with wild-type mice as well as in wild-type mice treated with the parp activity inhibitors tiq-a  <cit> , although pharmacological inhibition of parp activity using the pj <dig> inhibitor in peripheral blood lymphocytes did not suppress il- <dig> production  <cit> .
these discrepancies might be associated to side effects of the different inhibitors used in these studies.
further studies are therefore needed to determine whether parp- <dig> is a new player in the epigenetic regulation of il- <dig> by t cells  <cit>  or is involved on the immune response associated with a th <dig> bias.
autoinmunity seem to be associated to an immune response bised to the differentiation and activation of th <dig> and/or th <dig> effectors t cells.
parp- <dig> seems to positively control ifi <dig> expression even in the absence or presence of cd28-stimulation .
in sharp contrast, ifi202b was preferentially expressed in parp-1-/- stimulated t cells.
a high expression of ifi202b and a low expression of ifi <dig> and was also observed in b <dig> nba <dig> autoimmune prone mice  <cit> .
therefore, parp- <dig> could play a role in the consequence of autorecognition acting through ifi <dig> and ifi202b gene regulation.
t cell proliferation is impaired in the absence of parp-1
t cell proliferation is a hallmark of t cell activation.
to determine whether parp-1-dependent transcriptional regulation influences t cell function, we analyzed the proliferation response of parp-1-/- and control t cells after in vitro stimulation with anti-cd <dig> mab alone or in combination with anti-cd <dig> mab.
figure 5a shows a modest, but statistically significant, decrease in t cell proliferation upon stimulation in parp-1-/- compared to control cells while t cells from both genotypes show similar susceptibility to death upon activation suggesting an intrinsic defect in proliferation in the absence of parp- <dig> .
the role of parp- <dig> in apoptosis is variable, depending on the stimuli and the type of cells.
oliver et al.
reported that primary bone marrow parp-1-/- cells are extremely sensitive to apoptosis induced by an alkylating agent but not by a topoisomerase i inhibitor cpt- <dig> or by interleukin- <dig> removal  <cit> .
however, wang et al.
reported that parp-1-/- cells lymphoid cells display normal apoptotic sensitivity after treatment with various apoptotic agents  <cit> .
in addition, recently we have demonstrated that parp- <dig> deficiency affects cd4+cd8+ apoptosis susceptibility while we did not found any effect on parp-1-/- mice  <cit> .
although proliferation is a complex process that requires modulation of different genes, some of the parp- <dig> dependent genes identified in this analysis may help to understand the mutant phenotypes observed, thereby establishing a possible link between the transcriptome and the proliferation phenotype.
thus, the reduced expression of slamf <dig> and rel  <cit>  genes in the absence of parp- <dig> might be related with the reduced proliferative response of parp-1-/- t cells.
CONCLUSIONS
this study provides new insights into the role of parp- <dig> in t cell activation by modulating gene expression.
using a microarray based approach, we identified  <dig> genes whose expression in response to anti-cd <dig> stimulation is parp- <dig> dependent.
likewise, expression of  <dig> genes upon anti-cd <dig> + anti-cd <dig> stimulation was also dependent of parp- <dig>  by using automatic functional annotation of parp-1-dependent genes we identified go terms that were significantly over-represented in each activation condition , including a group of genes involved in lymph immune response.
functional dissection should help to unravel the remaining mysteries of parp- <dig> as a signalling molecule at the nuclear level in t cells and might be essential for future development of new therapeutic approaches targeting parp- <dig> in the acquired immune response.

 t lymphocyte activation requires recognition through the antigen-specific t cell receptor /cd complex of foreign peptides presented by self-mhc molecules on antigen presenting cells in context with co-stimulatory signals mediated by co-stimulatory receptors such as cd. the concurrent ligation of these receptors initiates signal transduction pathways that lead to the induction of a complex array of kinases and phosphatases, and the downstream activation of transcription factors such as nf-κb, ap- and nfat which play a critical role in reprogramming gene expression. the overall result is the proliferation, differentiation and activation of t cells. however, t cells stimulated via the tcr/cd complex alone do not become fully activated and can become anergic or even apoptotic. transcriptional changes during t cell stimulation are tightly regulated by a variety of mechanisms involving not only interactions between a complex network of transcription factors and cis-regulatory dna regions, but also epigenetic changes in chromatin structure including acetylation, phosphorylation, methylation and ubiquitylation. accumulating evidence suggests an important role for the enzyme poly polymerase- in the regulation of gene expression during development and in response to specific cellular signals working at different levels. parp- belongs to a family of enzymes that, using nad+ as a substrate, synthesize and transfer homopolymers of adp-ribose onto glutamic acid residues of acceptor proteins mainly involved in chromatin structure and dna metabolism. polyation is terminated by the release of extensively polyated parp molecules from dna. adp-ribose polymers are then subjected to degradation by poly glycohydrolase. polyation is therefore an immediate, covalent, but transient post-translational modification of cellular proteins playing and important role in epigenetic regulation of chromatin structure and gene expression under physiological conditions in which the integrity of the dna is maintained. parp- activity, responsible for almost 90% of polylation in the cell, might modulate gene expression through polyation of its partner proteins or by its physical association with relevant proteins such as transcription factors. regulation by parp- has been described for different transcription factors such as nf-κb, e2f-, c/ebpalpha, yy-, rna polymerase ii-associated factors, p and nfat. indeed, parp- and polyation play a critical role in the expression control of multiple nf-κb dependent genes involved in the inflammatory response. in addition, different studies have analysed the effects of parp- deficiency on gene expression in fibroblasts, cardiomyocytes, leukemia cells, glia, endothelial cells, embryonic stem cell lines and liver tissue at the genome-wide level. recently, we have demonstrated that parp- is activated during t cell activation, modulating the activity of the nfat transcription factor. in the present study we use oligonucleotide microarray analysis to gain more insight into the role played by parp- during the gene expression reprogramming that takes place in t cells upon activation with anti-cd3-stimulation alone, or in combination with anti-cd co-stimulation. these two stimulation conditions would allow us to analyse the role of parp- in the modulation of genes upon nfat activation under conditions in which both ap- and nf-κb are fully activated or poorly activated. t cells co-stimulated with anti-cd + anti-cd develop an overlapping transcription network with anti-cd stimulated t lymphocytes, but co-stimulatory signalling quantitatively alters components of the tcr/cd complex-induced signals. therefore, parp- could synergize with different transcription factors in order to stimulate or repress gene expression in both stimulation conditions. we have identified several groups of genes, whose expression is modulated by parp- either in a positive or a negative manner. interestingly, parp- deficiency significantly alters expression of genes associated with the immune response such as chemokines and genes involved in the th1/th balance.  transcriptional programs in parp-1-/- vs parp-1+/+ t cells in response to antigen receptor stimulation in order to study the role of parp- in the transcriptional regulation of t cells, we have analysed the gene expression profiles in splenic t cells derived from parp-1-wild-type and parp-1-deficient mice under both resting and antigen receptor stimulation conditions, using oligonucleotide microarray technology. total rna prepared from resting or stimulated cells was purified, reverse transcribed and amplified by in vitro transcription in the presence of biotinylated nucleotides, and the labeled crna was hybridized on affymetrix oligonucleotide chips containing over probe sets representing transcripts from over well-characterized mouse genes. only those differences in rna abundance that were reproducible in independent experiments with different batches of cells and represented by a change of -fold or greater were considered. at a basal level, we found genes that differed in their expression between parp-1+/+ and parp-1-/- t cells. this percentage is quite similar to the change in the basal expression level observed between primary parp-1+/+ and parp-1-/- fibroblasts. the expression of of these genes in t cells was higher in parp-1-/- than in parp-1+/+ t cells, while the expression of genes, obviously including the parp- gene itself, was lower in parp-1-/- compared to parp-1+/+ t cells. the specific genes that were differentially expressed in parp-1-/- vs parp-1+/+ t cells are listed in the additional files and in addition, all data are deposited in the arrayexpress database with accession number e-mexp- we did not observe genes overlapping between the report on primary fibroblast and our present results. similar absence of overlap has been reported recently by ogino et al, between fibroblasts and es cells. this may be possibly due to differences in the cell linages examined in these different experiments. a genes were classified as parp- dependent genes when their expression was change by a factor of at least between parp-1+/+ and parp-1-/- cells in the microarrays analysis. bpositive regulation was considered when the expression of a particular gene was higher in the presence of parp- than in its absence. likewise, genes expressed at higher level in the absence of parp- than in its presence were considered negatively regulated by parp- to characterize the gene expression program in t cells responding to antigen receptor stimulation, t cells from both genotypes were subjected to stimulation for h with a plate-bound antibody to cd alone, or in combination with an antibody to cd and expression results were normalized to the unstimulated conditions. short stimulation times have been shown to induce important changes in t cells gene expression programs. cd stimulation modulated a significant number of early-response genes in parp-1+/+ t cells, up-regulating and concomitantly down-regulating transcripts, while in parp-1-/- t cells of genes were modulated, up-regulating genes and down-regulating transcripts. on the other hand, cd stimulation in the context of cd co-stimulatory signal, modulated a significant number of early-response genes in parp-1+/+ t cells, up-regulating and concomitantly down-regulating transcripts, while in parp-1-/- t cells of genes were modulated, up-regulating genes and down-regulating transcripts. identification of parp-1-dependent genes during early t cell activation transcriptional changes during t lymphocyte activation have also been characterized in the past, and it is beyond the scope of this paper to provide a discussion of all of the observed changes and their biological implications. however, comparison of the gene expression programs evoked by activation in parp-1+/+ and parp-1-/-t cells shows that early-response genes to anti-cd seem to be parp-1-dependent and regulated by parp- either in a positive or in a negative manner. likewise, in the presence of co-stimulation, the expression of genes is also regulated by parp- either in a positive or in a negative manner. in addition, these data are listed in the additional files and interestingly, response to anti-cd alone resulted in a higher number of genes negatively regulated by parp- than those regulated positively. however, anti-cd stimulation in the presence of anti-cd co-stimulation produces the opposite effect, favouring expression of genes regulated positively by parp-. these results could be related with a different role of parp- in the regulation of specific transcription factors involved in those signalling pathways. thus, t cell stimulation via the tcr/cd complex alone seem to be sufficient for nfat but not for optimal nf-κb or ap- activation. we have recently demonstrated that parp- inhibition seems to modulate in an opposite manner nfat and nf-κb-dependent transcriptional activity in t cells, improving nfat and inhibiting nf-κb activities. however, the complexity of the nfat and nf-κb families, together with the regulation of genes by both transcriptional factor members precludes ascribing the described parp-1-dependent genes to a unique and direct effect on nfat or nf-κb dependent transcriptional activity. twenty one genes were dependent on parp- for response to both anti-cd and anti-cd + anti-cd stimulation. eleven of these genes were positively regulated by parp- while of them were negatively regulated by parp- in response to either anti-cd or anti-cd + anti-cd stimulation. however, genes were regulated positively by parp- in response to cd stimulation alone, but the same genes were regulated negatively by parp- in the presence of co-stimulation through cd likewise, genes were regulated negatively by parp- in response to cd stimulation but positively by parp- in response to anti-cd + anti-cd stimulation. we confirmed expression changes for some genes by real-time pcr. although there were differences in the absolute fold-change values detected by the two methods, pcr results correlated well with the differential gene expression data produced using affymetrix genechips. automatic functional annotation of parp-1-dependent genes in response to either anti-cd or anti-cd + anti-cd stimulation was performed based on ingenuity software. although this type of annotation is very useful to obtain a description of the transcriptome, it can be misleading if the absolute number of genes that fall into each class is used as the only factor for selection of relevant classes. consequently, we identified which gene-ontology terms were significantly over-represented in each activation condition. as shown in the examples in figure more parp-1-dependent genes than expected by chance were involved in certain processes or functions. the bars indicate whether there are more parp-1-dependent genes with a given go term than expected with a random distribution in each activation condition. interestingly, the highest differences were found for genes involved in lymphocyte activation and immune response, with little differences in genes involved in metabolism. parp- deficiency affects the expression of genes involved in the immune response pathway in t cells a subset of parp- dependent genes in response to anti-cd stimulation and in response to anti-cd + anti-cd stimulation encode for molecules involved directly in the immune response pathway as determined using the ingenuity software. to validate the array data, we also evaluated expression of several of these genes by quantitative real-time pcr. although there were differences in the absolute fold-change values detected by the two methods, pcr results correlated well with the differential gene expression data produced using affymetrix genechips. discordant results were found between the arrays and pcr for only gbp gene in response to anti-cd + anti-cd stimulation. in response to stimulation through cd alone or in combination with mab to cd a significantly increased expression of il- was detected in parp-1+/+ compared to parp-1-/- t cells. this result was further confirmed by real-time pcr. in addition, t cells purified from parp-1+/+ and parp-1-/- mice were stimulated for hours with anti-cd alone or in combination with anti-cd mab, and il- released in the supernatants was measured by elisa. the results verified the effect of parp- deficiency on the expression levels of il- obtained at the mrna level in the microarray and real-time pcr experiments. overall, our data suggest an important role for parp- in the regulation of il- expression. il- is a major th effector cytokine and a key promoter of th development. th and th cells are characterized by the types of cytokines that they produced. ifn-gamma is the hallmark of th cells which are involved in the immune response against intracellular microoganisms. on the other hand, th effector cells secrete il- il- and il- being involved in immune response to helminth infections as well as to ige-dependent hypersensitivity reactions. interestingly, the expression of ifn-gamma mrna seem to be increased in parp-1-/- t cells. expression of several th chemokines such as xcl ccl and ccl seem to be also increased en parp-1-/- t cells in response to anti-cd + anti-cd activation. therefore, the absence of parp- seems to bias t cell response to a th phenotype. however, our microarray data did not reveal any difference in the expression of other th cytokines such as il- il- and il- between parp-1-/- and parp-1+/+ t cells, suggesting that parp- could specifically control il- gene expression which might act on th differentiation by supressing il-12br expression and ifn-gamma secretion. previous studies have demonstrated the requirement of slam in il- production by t cells. similarly, irf gene expression was also found to be higher in parp-1+/+ than in parp-1-/- t cells after stimulation with anti-cd + anti-cd. irf seems to enhance il- but not il- promoter activity. whether parp- controls il- expression through an interaction with cis-regulatory elements or chromatin remodelling at il- locus remains to be elucidated. reduction of il- production by lung cells has been reported in ova-challenged parp-1-/- mice compared with wild-type mice as well as in wild-type mice treated with the parp activity inhibitors tiq-a, although pharmacological inhibition of parp activity using the pj inhibitor in peripheral blood lymphocytes did not suppress il- production. these discrepancies might be associated to side effects of the different inhibitors used in these studies. further studies are therefore needed to determine whether parp- is a new player in the epigenetic regulation of il- by t cells or is involved on the immune response associated with a th bias. autoinmunity seem to be associated to an immune response bised to the differentiation and activation of th and/or th effectors t cells. parp- seems to positively control ifi expression even in the absence or presence of cd28-stimulation. in sharp contrast, ifi202b was preferentially expressed in parp-1-/- stimulated t cells. a high expression of ifi202b and a low expression of ifi and was also observed in b nba autoimmune prone mice. therefore, parp- could play a role in the consequence of autorecognition acting through ifi and ifi202b gene regulation. t cell proliferation is impaired in the absence of parp-1 t cell proliferation is a hallmark of t cell activation. to determine whether parp-1-dependent transcriptional regulation influences t cell function, we analyzed the proliferation response of parp-1-/- and control t cells after in vitro stimulation with anti-cd mab alone or in combination with anti-cd mab. figure 5a shows a modest, but statistically significant, decrease in t cell proliferation upon stimulation in parp-1-/- compared to control cells while t cells from both genotypes show similar susceptibility to death upon activation suggesting an intrinsic defect in proliferation in the absence of parp-. the role of parp- in apoptosis is variable, depending on the stimuli and the type of cells. oliver et al. reported that primary bone marrow parp-1-/- cells are extremely sensitive to apoptosis induced by an alkylating agent but not by a topoisomerase i inhibitor cpt- or by interleukin- removal. however, wang et al. reported that parp-1-/- cells lymphoid cells display normal apoptotic sensitivity after treatment with various apoptotic agents. in addition, recently we have demonstrated that parp- deficiency affects cd4+cd8+ apoptosis susceptibility while we did not found any effect on parp-1-/- mice. although proliferation is a complex process that requires modulation of different genes, some of the parp- dependent genes identified in this analysis may help to understand the mutant phenotypes observed, thereby establishing a possible link between the transcriptome and the proliferation phenotype. thus, the reduced expression of slamf and rel genes in the absence of parp- might be related with the reduced proliferative response of parp-1-/- t cells.  this study provides new insights into the role of parp- in t cell activation by modulating gene expression. using a microarray based approach, we identified genes whose expression in response to anti-cd stimulation is parp- dependent. likewise, expression of genes upon anti-cd + anti-cd stimulation was also dependent of parp- by using automatic functional annotation of parp-1-dependent genes we identified go terms that were significantly over-represented in each activation condition, including a group of genes involved in lymph immune response. functional dissection should help to unravel the remaining mysteries of parp- as a signalling molecule at the nuclear level in t cells and might be essential for future development of new therapeutic approaches targeting parp- in the acquired immune response. 
accumulating evidence suggests an important role for the enzyme poly polymerase- as an integral part of the gene expression regulatory machinery during development and in response to specific cellular signals. interestingly, parp- deficiency significantly alters expression of genes associated with the immune response such as chemokines and genes involved in the th1/th balance. this study provides new insights into changes in gene expression mediated by parp- upon t cell activation. pathway analysis of parp- as a nuclear signalling molecule in t cells would be of relevance for the future development of new therapeutic approaches targeting parp- in the acquired immune response. parp- might modulate gene expression through its catalytic activity leading to polyation of nuclear proteins or by its physical association with relevant proteins. recently, we have shown that parp- is activated during t cell activation. in the present study we use oligonucleotide microarray analysis to gain more insight into the role played by parp- during the gene expression reprogramming that takes place in t cells upon activation with anti-cd stimulation alone, or in combination with anti-cd co-stimulation. however, the proposed role of parp- in reprogramming t cell gene expression upon activation remains largely unexplored. we have identified several groups of genes with expression modulated by parp- the expression of early-response genes to anti-cd seems to be regulated by parp- either in a positive or in a negative manner.
 accumulating evidence suggests an important role for the enzyme poly polymerase- as an integral part of the gene expression regulatory machinery during development and in response to specific cellular signals. parp- might modulate gene expression through its catalytic activity leading to polyation of nuclear proteins or by its physical association with relevant proteins. recently, we have shown that parp- is activated during t cell activation. however, the proposed role of parp- in reprogramming t cell gene expression upon activation remains largely unexplored.  in the present study we use oligonucleotide microarray analysis to gain more insight into the role played by parp- during the gene expression reprogramming that takes place in t cells upon activation with anti-cd stimulation alone, or in combination with anti-cd co-stimulation. we have identified several groups of genes with expression modulated by parp- the expression of early-response genes to anti-cd seems to be regulated by parp- either in a positive or in a negative manner. likewise, in the presence of co-stimulation, the expression of genes is also regulated by parp- either up or down. interestingly, parp- deficiency significantly alters expression of genes associated with the immune response such as chemokines and genes involved in the th1/th balance.  this study provides new insights into changes in gene expression mediated by parp- upon t cell activation. pathway analysis of parp- as a nuclear signalling molecule in t cells would be of relevance for the future development of new therapeutic approaches targeting parp- in the acquired immune response. 