BACKGROUND
t cells are among the most versatile cells in the body and play a central role in adaptive immunity.
t-cell maturation in thymus is a stepwise process, undergoing positive and negative selection to produce cd4+ and cd8+ t cells  <cit> .
when mature, t lymphocytes leave the thymus and are considered naive cells until they encounter activating signals in peripheral lymphoid organs, thus become activated, start to proliferate, differentiate into effector cells  and gain the ability to enter inflammations sites  <cit> .
activation of the naive t cells in the peripheral immune system is the first step of the adaptive immune response.
successful t-cell activation requires two major stimulatory signals to produce an effective immune response.
first, the t-cell receptor complex  recognizes the cognate ligands presented by the major histocompatibility complex  on antigen-presenting cells   <cit> .
second, a co-stimulation signal is presented to t cells through the engagement of a co-receptor such as cd <dig>  <cit> .
in the absence of cd <dig> co-stimulation, tcr signalling alone results in anergy.
t-cell activation, which begins with tcr activation with cd <dig> co-stimulation, triggers multiple signalling pathways and cellular events.
signalling downstream of tcr engagement has been widely studied  <cit> .
key events include activation of protein kinases such as lck and zap <dig>  intracellular ca2+ regulation, activation of map-kinase cascades, and activation and nuclear localization of crucial transcription factors including ap- <dig>  nfat, and nf-κb.
however, our understanding of the activation process including subsequent proliferation and differentiation events is far from complete.
a temporal genome-scale transcription profiling of t-cell activation process would provide a comprehensive understanding and insights into the molecular mechanisms underlying the process.
gene expression analysis of t-cell activation at a single timepoint has been reported  <cit> , and using a single donor sample, the gene expression patterns of t-cell activation with or without co-stimulation by anti-cd <dig> antibody were compared  <cit> .
however, to the best of our knowledge, the genome-scale donor-independent temporal gene expression analysis of primary, human t-cell activation has not been reported, and this is the goal of this study.
significantly, a comparative analysis of the programs of two t-cell subsets  against each other and against the natural cd3+ population remains unexplored, and would likely yield significant new information.
comparison of the transcriptional patterns among the three populations should lead to the identification of the common transcriptional events shared by cd4+ and cd8+ t cells, and of subset-specific genes and genes potentially involved in the communication between cd4+ and cd8+ t cells.
among the differentially expressed genes, we focused on 'immune response' genes based on gene ontology  classification in order to provide new insights into the expression of chemokines and cytokines, the orchestrated regulation of receptors, the interactions between the two subsets, and the homeostasis of resting t cells.
such understanding would be helpful for enhancing, re-directing or modifying the activities of t cells under physiological and pathophysiological circumstances.
RESULTS
primary human t-cell activation is donor independent
we aimed to capture important, donor-independent transcriptional events of t-cell activation.
three biological experiments, e1–e <dig>  using cd3+ t cells, which contain both the cd4+ and cd8+ subsets, from three different healthy donors demonstrated similar phenotypic characteristics.
t-cell proliferation, as measured by cell expansion, started at  <dig> hours after stimulation, and cell numbers doubled by  <dig> hours .
cell viability remained around 80% throughout the  <dig> hours .
surface expression of the early t-cell activation marker cd <dig> was rapidly upregulated within  <dig> hours, and then downregulated after  <dig> hours .
expression of the other important surface marker cd <dig>  rapidly increased within  <dig> hours and stayed high  from  <dig> hours to  <dig> hours .
we also examined the cd4+/cd8+ subset ratio but we found no significant changes during the  <dig> hours of the experiments .
cd4+ cells were ca.
60%, and cd8+ cells ca.
40% of the total t-cell population.
a separate set of experiments, e7–e <dig>  was carried out using separately cd4+ and cd8+ t cells isolated from another three healthy donors.
the time course analysis of this set of experiments was setup somewhat differently than in the cd3+ t-cell study  in order to cover earlier timepoints.
as we demonstrate below, the different time points in the two sets of experiments do not affect our ability to compare the data from the two studies, and in fact enhance and broaden the validity of the conclusions.
within each population, t cells exhibited overall similar phenotypic characteristics .
t-cell proliferation as assessed by cell numbers did not start until  <dig> hours.
expansion reached about  <dig>  fold in cd4+ t cells and about  <dig>  fold in cd8+ t cells by  <dig> hours.
cell viability remained around 75% in cd4+ t cells vs. around 60% in cd8+ t cells; the lower viability of cd8+ t cells was likely caused by the absence of help from cd4+ t cells.
expression of the t-cell activation surface markers cd <dig> and cd <dig> in the two subsets was similar to that of cd3+ t cells.
agilent microarrays that target  <dig>  human genes were used to generate the transcriptional profile of activation for the cd3+ t-cell population, and the cd4+ and cd8+ t-cell subsets.
comparing samples across all time points, multi-class sam  identified  <dig> genes with statistically significant expression changes in the cd3+ population,  <dig> significant genes in the cd4+ population, and  <dig> significant genes in the cd8+ population.
hierarchical clustering  demonstrated that the transcriptional patterns of these significant genes among the replicate biological experiments within each population were highly reproducible.
thus, for simplicity and ease of presentation, gene expression data from the three biological experiments for each population were averaged for discussion and analysis below.
in order to compare the transcriptional patterns of cd3+, cd4+ and cd8+ t cells, the significant genes from all experiments were combined to a total of  <dig> unique, significant genes distributed among the  <dig> t-cell populations as shown in the venn diagram of figure  <dig>  far more significant genes were identified in the cd3+ t-cell activation experiments than in the subset experiments, possibly reflecting a larger repertoire of genes during activation in the natural, mixed population of cd3+ t cells, and thus the synergy and interplay of the two subsets  in producing a more complex and multifaceted response.
nevertheless, a large number of the genes were shared by cd4+ and cd8+ t cells , reflecting the common cellular events shared by cd4+ and cd8+ t cells during the activation process.
hierarchical clustering of these pooled significant genes  demonstrated that the  <dig> populations shared largely similar transcriptional profiles regardless of the difference of the sampled timepoints, which, in perspective, broadens the significance of identified genes.
q-rt-pcr was used to validate select microarray results.
fifteen significant genes with different expression intensities were selected.
as previously reported  <cit> , in our laboratory, data from these agilent microarrays correlated strongly with the q-rt-pcr results, although q-rt-pcr data generally show larger fold changes compared to microarray data .
we thus conclude that the t-cell activation process under our experimental conditions is largely donor invariant, as assessed by both phenotypic data and transcriptional profiles.
regulation of ' immune response' genes in t-cell activation
ontological analysis using the mev ease module identified  <dig> genes associated with the term 'immune response' among the  <dig> significant genes, consistent with the essential roles of t cells in the adaptive immune response.
hierarchical clustering revealed distinct expression patterns for these  <dig> genes and allowed us to divide them into two clusters:  expression is mainly upregulated compared to resting t cells  ;  expression is mainly downregulated compared to resting t cells  .
although we expected differences in gene expression patterns because of the different biology and functions of cd4+ and cd8+ t cells, the aforementioned  <dig> genes show, overall, similar expression patterns among the  <dig> populations but with several notable exceptions.
within the upregulated cluster a, notable differences among the three populations include:  genes in clusters i  and iv , which shared similar gene expression patterns between cd4+ and cd8+ t cells, but different than those of cd3+ t cells;  genes in cluster ii ) with different gene expression patterns among the  <dig> populations, and cluster iii  with different gene expression patterns between the cd4+ and cd8+ populations.
within the downregulated cluster b, there were a few genes with different expression patterns in cluster v , mainly between cd4+ and cd8+ t cells, and cluster vi , mainly between cd3+ t cells and cd4+, cd8+ subsets.
genes sharing similar expression patterns between cd4+ and cd8+ t cells, but different than cd3+ t cells, are likely important players in the communication between cd4+ and cd8+ compartments.
although not previously associated with t-cell activation, the decreased upregulation of nr4a <dig>   <cit>  and increased upregulation of ifi <dig>   <cit>  and psmb <dig>  psme <dig>  and otub <dig>  <cit>  are possibly involved in the delayed t-cell activation and proliferation of the cd4+ and cd8+ subsets compared to cd3+ t cells.
the significant downregulation  of igll <dig> and igj , fcer1a , cd <dig>  and irf <dig>  in cd3+ t cells, but not in cd4+ or cd8+ subset suggests that these proteins are affected by the co-presence and/or communication between the two subsets.
genes with different expression patterns between cd4+ and cd8+ subsets are possibly involved in cell-type-specific characteristics and functions.
for instance, expression of tnfsf <dig>  has been mainly reported in cd4+ t cells, facilitating the activation of cd8+ t cells  <cit> .
indeed, a preferential transcriptional upregulation of tnfsf <dig> was observed in cd4+ t cells, supported by a protein abundance assay .
interestingly, tnfsf <dig> was also upregulated in cd8+ t cells , supporting the recently reported expression of tnfsf <dig> in cd8+ t cells in the absence of cd4+ t cells  <cit> .
this is an example demonstrating that, with the comparative analysis of the expression patterns among the  <dig> populations, our data capture significant differential transcriptional events.
transcriptional differences between cd4+ and cd8+ subsets were validated and supported by protein abundance assays of selected genes  .
some of these genes  are discussed in detail below.
cytokines act as messengers between cells, regulating their functions and activity.
the production of cytokines is precisely controlled temporally in the immune response, and so are cytokine receptors.
thus, the significantly regulated cytokines and cytokine receptors in t-cell activation were sorted based on their functions listed by ncbi  <cit>   and are discussed below.
cytokines and chemokines
a group of chemokines  showed a steady increasing expression upon anti-cd3/anti-cd <dig> stimulation in all  <dig> populations with the exception that xcl <dig> was not upregulated in cd4+ t cells.
the strong upregulation of these genes is likely responsible for the proinflammatory response of t cells, including the recruitment of t cells as well as other leukocytes to the sites of inflammation.
ccl <dig> is mainly secreted by epithelial cells and macrophages  <cit> , and cxcl <dig>  - <dig>  - <dig> are mainly secreted by dendritic cells and macrophages  <cit> ; their expressions have not been reported in t cells.
supernatant elisa assays confirmed the significant continuous transcription upregulation of ccl <dig> , suggesting the induction of ccl <dig> secretion in t-cell activation.
the preferential expression of xcl <dig> and xcl <dig> in cd8+ t cells suggests that these proteins might have roles in activation and/or functions of cytotoxic t cells  <cit> .
a few chemokines showed high expression in resting t cells , suggesting their importance in the homeostasis of resting t cells in the peripheral immune system.
interferon gamma , infg-inducible protein  <dig> , ifn regulatory factor  <dig> , - <dig>  and ifng-inducible guanylate binding proteins  were all upregulated .
supernatant elisa assays revealed that the secreted ifng protein level continuously increased throughout the  <dig> hours .
ifng has important immunoregulatory functions such as antiviral and anti-tumor activity, and as an activator of macrophages  <cit> , yet its functions in t-cell activation remains unknown.
the orchestrated transcriptional regulation of ifn regulatory factors, ifng, and infg-inducible proteins and the significant induction of ifng protein secretion suggest that ifng secreted by t cells has an important role in t-cell activation.
some of the interleukins  were also upregulated, and with distinct patterns.
the expression pattern of il23a in cd3+ t cells was significantly different than that in cd4+ or cd8+ t cells.
expressed by activated dendritic cells, il <dig> has anti-tumor effects through inducing cd4+ t-cell proliferation and its anti-tumor effects are reportedly to be inhibited by the depletion of cd4+ or cd8+ subset  <cit> .
these expression differences among the three populations suggest that il23a might be implicated in the communication between cd4+ and cd8+ t cells.
early upregulation of il <dig>   <cit>  and il <dig>   <cit>  suggests that they might have important roles in t cells as activating/differentiation factors.
the downregulation  of il1b, mainly secreted by macrophages  <cit> , and il <dig>  an important cytokine in lymphocyte survival  <cit> , upon anti-cd3/anti-cd <dig> stimulation was unexpected.
a few members of the tnf family , - <dig>  - <dig> and tnf) were upregulated while others  and tnfsf12-tnfsf13) were downregulated.
a preferential expression in cd8+ t cells especially at  <dig> hours was observed for tnfsf <dig>  tnfsf <dig> has been hypothesized to have costimulatory functions in both cd4+ and cd8+ subsets  <cit> .
however, no subset-specific functions of tnfsf <dig> have been reported.
flow-cytometric analysis confirmed its significant upregulation at  <dig> hours in cd8+ t cells .
this preferential expression suggests that in cd8+ t cells, tnfsf <dig> might play an important role, possibly with cytotoxic effector functions besides the reported costimulatory functions.
a few cytokines  also displayed increased expression mainly at 10– <dig> hours.
neither the expression nor the function of these genes has been previously reported in t cells.
receptors
the upregulation of several key receptors , icos, and il21r) was expected and confirms the validity of our data.
however, the temporal expression patterns of these upregulated receptors, including those expressed throughout these experiments , adora2b), early  or late , provide new insights that reflect their roles in t-cell activation.
for instance, the late upregulation of clta <dig> is consistent with its inhibitory functions in t-cell activation  <cit> .
the upregulation of fas at 4– <dig> hours implies that fas might have facilitating functions in early t-cell activation in addition to its known role in inducing apoptosis in fully activated cells  <cit> .
the il <dig> receptor is critical for inducing the development of the th <dig> lineage of effector t cells  <cit> .
the simultaneous early upregulation of il4r and il <dig>  suggests that the th1/th <dig> balance might be biased towards the th <dig> direction in our experiments.
significant differential expression of receptors, which have not been reported in t cells, call for attention to their possible role in t-cell activation.
these include the upregulation of il1r <dig>  at  <dig> hours and the constant upregulation of adora2b .
extensive upregulation of tnfrsf <dig> was observed in cd8+ subsets through 6– <dig> hours, but not in cd3+ t cells or cd4+ t cells.
flow cytometry analysis supported this preferential expression of tnfrsf <dig> in cd8+ t cells at the protein level .
tnfrsf <dig> has been reported as a costimulatory receptor in t cells, but not in a subset specific manner  <cit> .
this continuous strong upregulation of tnfrsf <dig> in cd8+ t cells indicates its specific involvement in the activation, proliferation and differentiation of cytotoxic t cells.
transcription of a number of other receptors, not previously reported in the context of t-cell action, was downregulated, likely to help achieve an efficient t-cell activation.
these include il7r  with il2ra)  <cit> , ccr <dig>  tnfrsf <dig> and tnfrsf1a   <cit> , ccr <dig>   <cit>  and il6r   <cit> .
the unexpected downregulation of il2rg  and of integrins , the components of lfa- <dig> , was somewhat surprising and deserves detailed attention.
our data reveal the transcriptional dynamics of a few preciously reported upregulated cell-surface antigens including cd <dig>  <cit>   and slamf <dig>   <cit>  , and novel ones such as cd <dig>  and cd <dig> .
interestingly, cd <dig> showed increased expression upon stimulation in cd3+ t cells, decreased expression at 24– <dig> hours in cd4+ t cells, but no change in cd8+ t cells.
cd <dig> expressed on dendritic cells delivers costimulatory signals to t cells for activation  <cit> , and cd <dig> expressed on t cells has been hypothesized to be involved in t-cell activation with its detailed function yet to be defined  <cit> .
these apparently different expression patterns among the three populations indicate that cd <dig> might have different roles in cd4+ versus cd8+ subsets, and may be possibly implicated in the communication of the subsets.
in contrast to cd3+ or cd4+ t cells, slamf <dig> showed significant increased expression in cd8+ t cells at 6– <dig> hours.
slamf <dig> regulates the cytotoxity of nk cells  <cit> , and migration/adhesion of b cells  <cit> ; however, its function in t cells has not been reported.
this preferential expression of slamf <dig> in cd8+ t cells suggests that it might be involved in the development of the cytotoxicity of cd8+ t cells.
among the mainly downregulated cell-surface antigens, the simultaneous downregulation of cd3d, cd3g, trat <dig>  and tyrobp  along with the downregulation of lck and zap <dig>  are likely part of the orchestrated regulation of t-cell activation.
cd <dig>  cd <dig>  and cd <dig> shared similar expression patterns .
however, little is known about their roles in t-cell activation.
we also observed the downregulation of cd8b and cd8a in cd8+ t-cell, possibly as a part of the cd8+ t-cell activation machinery.
interesting, binding partners, cell-surface antigens cd <dig> and cd <dig>  demonstrated opposite expression patterns.
cd <dig> was continuously downregulated in cd4+ t cells, but not in cd8+ t cells; while cd <dig> was continuously downregulated in cd8+ t cells, but not in cd4+ t cells.
it has been hypothesized that t cells costimulate each other through the interactions of cd <dig> and cd <dig>  <cit> .
this cell-type-specific downregulation of cd <dig>  and cd <dig>  suggests that this cd244–cd <dig> interaction might be cell-type-specific, between the cd <dig> expressing cd8+ t cells and cd <dig> expressing cd4+ t cells.
upon activation, killer cell lectin-like receptors  showed mainly decreased expression.
first discovered in nk cells, the expression of inhibitory receptors   <cit> , and activating receptor klrc <dig>  <cit>  has also been reported in activated cd8+ t cells, involved in tcr signalling, but not in activated cd4+ t cells or naive t cells.
the shared downregulation of these klrs along with that of some less-well-studied members  suggests that their function might not be limited to effector cd8+ t cells, and that they are implicated more broadly in t-cell activation.
surprisingly, members of both mhc classes i and ii were expressed in resting t cells .
several genes encoding mhc class ii members  showed decreased expression upon stimulation, while some mhc class i molecules  were downregulated at 48– <dig> hours.
granzymes
secretion of cytotoxic granules is one of the major effector functions of cytotoxic t cells to induce apoptosis in target cells  <cit> .
perforins, granulysin and granzymes are the core components of the dense cytotoxic granules responsible for target cell lysis.
most of the granzyme genes , but not gnly and prf <dig>  were significantly regulated .
gzma and gzmk were considerably downregulated throughout the experiments.
it is likely that t cells have not acquired the full cytotoxic effector functions at this stage of the activation.
however, gzmb was transcriptionally upregulated in both cd4+ and cd8+ t cells, and this was supported by data from a protein abundance assay .
discussion
genome-scale transcriptional profiling can add significant new information for better understanding t-cell activation as an important biological process of the immune response.
previous efforts had examined t-cell activation at one single time point  <cit>  or addressed costimulatory signal effects using only a single experiment  <cit> .
using multiple donors, in this study we focused on the temporal, donor-independent gene expression patterns not only in the cd3+ t cells, but also in the cd4+ and cd8+ subsets.
we identified donor-independent significantly regulated genes in t-cell activation in cd3+ t cells , and the cd4+ and cd8+ subsets.
cd3+ t cells had far more significantly differentially expressed genes than cd4+ or cd8+ t cells .
regardless, the transcription profiles of the pooled significant genes in t-cell activation shared largely similar patterns among the three populations.
anti-cd3/anti-cd <dig> stimulation induced expression pattern changes of 'immune response' genes which are consistent with the important roles of t cells in the adaptive immune response.
not only well-known cytokines ) and cytokine receptors , cd <dig>  icos), but also numerous novel ones, for t-cell activation, were differentially expressed.
among the novel cytokines, the strongly upregulated chemokines  might have important roles in enhancing t-cell activation in addition to their functions in cell trafficking, while the downregulated ccl <dig>  il <dig> might rather be involved in maintaining the homeostasis of resting t cells.
a number of cell-surface receptors not previously associated with t-cell activation, were differentially expressed, including downregulated ccr <dig>  tnfrsf <dig>  tnfrsf1a, ccr <dig> and il6r, and upregulated cd <dig> and cd <dig>  a number of the receptors involved in tcr activation  along with lck, zap <dig> and tyrobp, were all simultaneously downregulated.
little is known about the expression and functions of klrs in resting t cells.
the downregulated klr receptors are likely involved in the homeostasis of resting t cells.
it is also possible that they regulate t-cell activation through tcr signalling.
surprisingly, both mhc class i and class ii molecules were transcriptionally expressed in resting t cells  , and mhc class ii molecules were significantly downregulated upon t-cell activation.
mhc class i and class ii molecules are receptors on apcs, but not t cells, for the activation of cd8+ and cd4+ t cells, respectively.
comparison of the expression patterns among the three populations provided further insights.
different expression patterns between cd4+ and cd8+ t cells  were observed, suggesting their subset specific involvement/functions in t-cell activation.
among these genes, very little is known about xcl <dig> and slamf <dig> in t cells.
genes  with similar expression patterns between cd4+ and cd8+ t cells, but different than those in cd3+ t cells are likely involved in the communication between cd4+ and cd8+ subsets.
these and the large number of novel genes in the context of t-cell activation that were identified in this study offer new research targets for a more complete understanding of t-cell activation.
CONCLUSIONS
our study captured novel temporal patterns of previously known but many novel, in the context of t-cell activation, genes ontologically classified under the term 'immune response'.
these patterns were reproducibly and robustly identified as donor independent, and were selectively confirmed by q-rt-pcr and protein-level assays.
comprehensively integrating previous knowledge, we identified novel significant genes associated with the immune response in t cells, as well as subset specific genes, and genes implicated in the communication between cd4+ and cd8+ t cells.
this study improves our understanding of the biology and the underlying regulation of t-cell activation in the natural cd3+ population, as well as in the cd4+ and cd8+ subsets.

 t cells are among the most versatile cells in the body and play a central role in adaptive immunity. t-cell maturation in thymus is a stepwise process, undergoing positive and negative selection to produce cd4+ and cd8+ t cells. when mature, t lymphocytes leave the thymus and are considered naive cells until they encounter activating signals in peripheral lymphoid organs, thus become activated, start to proliferate, differentiate into effector cells and gain the ability to enter inflammations sites. activation of the naive t cells in the peripheral immune system is the first step of the adaptive immune response. successful t-cell activation requires two major stimulatory signals to produce an effective immune response. first, the t-cell receptor complex recognizes the cognate ligands presented by the major histocompatibility complex on antigen-presenting cells. second, a co-stimulation signal is presented to t cells through the engagement of a co-receptor such as cd. in the absence of cd co-stimulation, tcr signalling alone results in anergy. t-cell activation, which begins with tcr activation with cd co-stimulation, triggers multiple signalling pathways and cellular events. signalling downstream of tcr engagement has been widely studied. key events include activation of protein kinases such as lck and zap intracellular ca2+ regulation, activation of map-kinase cascades, and activation and nuclear localization of crucial transcription factors including ap- nfat, and nf-κb. however, our understanding of the activation process including subsequent proliferation and differentiation events is far from complete. a temporal genome-scale transcription profiling of t-cell activation process would provide a comprehensive understanding and insights into the molecular mechanisms underlying the process. gene expression analysis of t-cell activation at a single timepoint has been reported, and using a single donor sample, the gene expression patterns of t-cell activation with or without co-stimulation by anti-cd antibody were compared. however, to the best of our knowledge, the genome-scale donor-independent temporal gene expression analysis of primary, human t-cell activation has not been reported, and this is the goal of this study. significantly, a comparative analysis of the programs of two t-cell subsets against each other and against the natural cd3+ population remains unexplored, and would likely yield significant new information. comparison of the transcriptional patterns among the three populations should lead to the identification of the common transcriptional events shared by cd4+ and cd8+ t cells, and of subset-specific genes and genes potentially involved in the communication between cd4+ and cd8+ t cells. among the differentially expressed genes, we focused on'immune response' genes based on gene ontology classification in order to provide new insights into the expression of chemokines and cytokines, the orchestrated regulation of receptors, the interactions between the two subsets, and the homeostasis of resting t cells. such understanding would be helpful for enhancing, re-directing or modifying the activities of t cells under physiological and pathophysiological circumstances.  primary human t-cell activation is donor independent we aimed to capture important, donor-independent transcriptional events of t-cell activation. three biological experiments, e1–e using cd3+ t cells, which contain both the cd4+ and cd8+ subsets, from three different healthy donors demonstrated similar phenotypic characteristics. t-cell proliferation, as measured by cell expansion, started at hours after stimulation, and cell numbers doubled by hours. cell viability remained around 80% throughout the hours. surface expression of the early t-cell activation marker cd was rapidly upregulated within hours, and then downregulated after hours. expression of the other important surface marker cd rapidly increased within hours and stayed high from hours to hours. we also examined the cd4+/cd8+ subset ratio but we found no significant changes during the hours of the experiments. cd4+ cells were ca. 60%, and cd8+ cells ca. 40% of the total t-cell population. a separate set of experiments, e7–e was carried out using separately cd4+ and cd8+ t cells isolated from another three healthy donors. the time course analysis of this set of experiments was setup somewhat differently than in the cd3+ t-cell study in order to cover earlier timepoints. as we demonstrate below, the different time points in the two sets of experiments do not affect our ability to compare the data from the two studies, and in fact enhance and broaden the validity of the conclusions. within each population, t cells exhibited overall similar phenotypic characteristics. t-cell proliferation as assessed by cell numbers did not start until hours. expansion reached about fold in cd4+ t cells and about fold in cd8+ t cells by hours. cell viability remained around 75% in cd4+ t cells vs. around 60% in cd8+ t cells; the lower viability of cd8+ t cells was likely caused by the absence of help from cd4+ t cells. expression of the t-cell activation surface markers cd and cd in the two subsets was similar to that of cd3+ t cells. agilent microarrays that target human genes were used to generate the transcriptional profile of activation for the cd3+ t-cell population, and the cd4+ and cd8+ t-cell subsets. comparing samples across all time points, multi-class sam identified genes with statistically significant expression changes in the cd3+ population, significant genes in the cd4+ population, and significant genes in the cd8+ population. hierarchical clustering demonstrated that the transcriptional patterns of these significant genes among the replicate biological experiments within each population were highly reproducible. thus, for simplicity and ease of presentation, gene expression data from the three biological experiments for each population were averaged for discussion and analysis below. in order to compare the transcriptional patterns of cd3+, cd4+ and cd8+ t cells, the significant genes from all experiments were combined to a total of unique, significant genes distributed among the t-cell populations as shown in the venn diagram of figure far more significant genes were identified in the cd3+ t-cell activation experiments than in the subset experiments, possibly reflecting a larger repertoire of genes during activation in the natural, mixed population of cd3+ t cells, and thus the synergy and interplay of the two subsets in producing a more complex and multifaceted response. nevertheless, a large number of the genes were shared by cd4+ and cd8+ t cells, reflecting the common cellular events shared by cd4+ and cd8+ t cells during the activation process. hierarchical clustering of these pooled significant genes demonstrated that the populations shared largely similar transcriptional profiles regardless of the difference of the sampled timepoints, which, in perspective, broadens the significance of identified genes. q-rt-pcr was used to validate select microarray results. fifteen significant genes with different expression intensities were selected. as previously reported, in our laboratory, data from these agilent microarrays correlated strongly with the q-rt-pcr results, although q-rt-pcr data generally show larger fold changes compared to microarray data. we thus conclude that the t-cell activation process under our experimental conditions is largely donor invariant, as assessed by both phenotypic data and transcriptional profiles. regulation of' immune response' genes in t-cell activation ontological analysis using the mev ease module identified genes associated with the term'immune response' among the significant genes, consistent with the essential roles of t cells in the adaptive immune response. hierarchical clustering revealed distinct expression patterns for these genes and allowed us to divide them into two clusters: expression is mainly upregulated compared to resting t cells; expression is mainly downregulated compared to resting t cells. although we expected differences in gene expression patterns because of the different biology and functions of cd4+ and cd8+ t cells, the aforementioned genes show, overall, similar expression patterns among the populations but with several notable exceptions. within the upregulated cluster a, notable differences among the three populations include: genes in clusters i and iv, which shared similar gene expression patterns between cd4+ and cd8+ t cells, but different than those of cd3+ t cells; genes in cluster ii ) with different gene expression patterns among the populations, and cluster iii with different gene expression patterns between the cd4+ and cd8+ populations. within the downregulated cluster b, there were a few genes with different expression patterns in cluster v, mainly between cd4+ and cd8+ t cells, and cluster vi, mainly between cd3+ t cells and cd4+, cd8+ subsets. genes sharing similar expression patterns between cd4+ and cd8+ t cells, but different than cd3+ t cells, are likely important players in the communication between cd4+ and cd8+ compartments. although not previously associated with t-cell activation, the decreased upregulation of nr4a and increased upregulation of ifi and psmb psme and otub are possibly involved in the delayed t-cell activation and proliferation of the cd4+ and cd8+ subsets compared to cd3+ t cells. the significant downregulation of igll and igj, fcer1a, cd and irf in cd3+ t cells, but not in cd4+ or cd8+ subset suggests that these proteins are affected by the co-presence and/or communication between the two subsets. genes with different expression patterns between cd4+ and cd8+ subsets are possibly involved in cell-type-specific characteristics and functions. for instance, expression of tnfsf has been mainly reported in cd4+ t cells, facilitating the activation of cd8+ t cells. indeed, a preferential transcriptional upregulation of tnfsf was observed in cd4+ t cells, supported by a protein abundance assay. interestingly, tnfsf was also upregulated in cd8+ t cells, supporting the recently reported expression of tnfsf in cd8+ t cells in the absence of cd4+ t cells. this is an example demonstrating that, with the comparative analysis of the expression patterns among the populations, our data capture significant differential transcriptional events. transcriptional differences between cd4+ and cd8+ subsets were validated and supported by protein abundance assays of selected genes. some of these genes are discussed in detail below. cytokines act as messengers between cells, regulating their functions and activity. the production of cytokines is precisely controlled temporally in the immune response, and so are cytokine receptors. thus, the significantly regulated cytokines and cytokine receptors in t-cell activation were sorted based on their functions listed by ncbi and are discussed below. cytokines and chemokines a group of chemokines showed a steady increasing expression upon anti-cd3/anti-cd stimulation in all populations with the exception that xcl was not upregulated in cd4+ t cells. the strong upregulation of these genes is likely responsible for the proinflammatory response of t cells, including the recruitment of t cells as well as other leukocytes to the sites of inflammation. ccl is mainly secreted by epithelial cells and macrophages, and cxcl - - are mainly secreted by dendritic cells and macrophages; their expressions have not been reported in t cells. supernatant elisa assays confirmed the significant continuous transcription upregulation of ccl, suggesting the induction of ccl secretion in t-cell activation. the preferential expression of xcl and xcl in cd8+ t cells suggests that these proteins might have roles in activation and/or functions of cytotoxic t cells. a few chemokines showed high expression in resting t cells, suggesting their importance in the homeostasis of resting t cells in the peripheral immune system. interferon gamma, infg-inducible protein, ifn regulatory factor, - and ifng-inducible guanylate binding proteins were all upregulated. supernatant elisa assays revealed that the secreted ifng protein level continuously increased throughout the hours. ifng has important immunoregulatory functions such as antiviral and anti-tumor activity, and as an activator of macrophages, yet its functions in t-cell activation remains unknown. the orchestrated transcriptional regulation of ifn regulatory factors, ifng, and infg-inducible proteins and the significant induction of ifng protein secretion suggest that ifng secreted by t cells has an important role in t-cell activation. some of the interleukins were also upregulated, and with distinct patterns. the expression pattern of il23a in cd3+ t cells was significantly different than that in cd4+ or cd8+ t cells. expressed by activated dendritic cells, il has anti-tumor effects through inducing cd4+ t-cell proliferation and its anti-tumor effects are reportedly to be inhibited by the depletion of cd4+ or cd8+ subset. these expression differences among the three populations suggest that il23a might be implicated in the communication between cd4+ and cd8+ t cells. early upregulation of il and il suggests that they might have important roles in t cells as activating/differentiation factors. the downregulation of il1b, mainly secreted by macrophages, and il an important cytokine in lymphocyte survival, upon anti-cd3/anti-cd stimulation was unexpected. a few members of the tnf family, - - and tnf) were upregulated while others and tnfsf12-tnfsf13) were downregulated. a preferential expression in cd8+ t cells especially at hours was observed for tnfsf tnfsf has been hypothesized to have costimulatory functions in both cd4+ and cd8+ subsets. however, no subset-specific functions of tnfsf have been reported. flow-cytometric analysis confirmed its significant upregulation at hours in cd8+ t cells. this preferential expression suggests that in cd8+ t cells, tnfsf might play an important role, possibly with cytotoxic effector functions besides the reported costimulatory functions. a few cytokines also displayed increased expression mainly at 10– hours. neither the expression nor the function of these genes has been previously reported in t cells. receptors the upregulation of several key receptors, icos, and il21r) was expected and confirms the validity of our data. however, the temporal expression patterns of these upregulated receptors, including those expressed throughout these experiments, adora2b), early or late, provide new insights that reflect their roles in t-cell activation. for instance, the late upregulation of clta is consistent with its inhibitory functions in t-cell activation. the upregulation of fas at 4– hours implies that fas might have facilitating functions in early t-cell activation in addition to its known role in inducing apoptosis in fully activated cells. the il receptor is critical for inducing the development of the th lineage of effector t cells. the simultaneous early upregulation of il4r and il suggests that the th1/th balance might be biased towards the th direction in our experiments. significant differential expression of receptors, which have not been reported in t cells, call for attention to their possible role in t-cell activation. these include the upregulation of il1r at hours and the constant upregulation of adora2b. extensive upregulation of tnfrsf was observed in cd8+ subsets through 6– hours, but not in cd3+ t cells or cd4+ t cells. flow cytometry analysis supported this preferential expression of tnfrsf in cd8+ t cells at the protein level. tnfrsf has been reported as a costimulatory receptor in t cells, but not in a subset specific manner. this continuous strong upregulation of tnfrsf in cd8+ t cells indicates its specific involvement in the activation, proliferation and differentiation of cytotoxic t cells. transcription of a number of other receptors, not previously reported in the context of t-cell action, was downregulated, likely to help achieve an efficient t-cell activation. these include il7r with il2ra), ccr tnfrsf and tnfrsf1a, ccr and il6r. the unexpected downregulation of il2rg and of integrins, the components of lfa-, was somewhat surprising and deserves detailed attention. our data reveal the transcriptional dynamics of a few preciously reported upregulated cell-surface antigens including cd and slamf, and novel ones such as cd and cd. interestingly, cd showed increased expression upon stimulation in cd3+ t cells, decreased expression at 24– hours in cd4+ t cells, but no change in cd8+ t cells. cd expressed on dendritic cells delivers costimulatory signals to t cells for activation, and cd expressed on t cells has been hypothesized to be involved in t-cell activation with its detailed function yet to be defined. these apparently different expression patterns among the three populations indicate that cd might have different roles in cd4+ versus cd8+ subsets, and may be possibly implicated in the communication of the subsets. in contrast to cd3+ or cd4+ t cells, slamf showed significant increased expression in cd8+ t cells at 6– hours. slamf regulates the cytotoxity of nk cells, and migration/adhesion of b cells; however, its function in t cells has not been reported. this preferential expression of slamf in cd8+ t cells suggests that it might be involved in the development of the cytotoxicity of cd8+ t cells. among the mainly downregulated cell-surface antigens, the simultaneous downregulation of cd3d, cd3g, trat and tyrobp along with the downregulation of lck and zap are likely part of the orchestrated regulation of t-cell activation. cd cd and cd shared similar expression patterns. however, little is known about their roles in t-cell activation. we also observed the downregulation of cd8b and cd8a in cd8+ t-cell, possibly as a part of the cd8+ t-cell activation machinery. interesting, binding partners, cell-surface antigens cd and cd demonstrated opposite expression patterns. cd was continuously downregulated in cd4+ t cells, but not in cd8+ t cells; while cd was continuously downregulated in cd8+ t cells, but not in cd4+ t cells. it has been hypothesized that t cells costimulate each other through the interactions of cd and cd. this cell-type-specific downregulation of cd and cd suggests that this cd244–cd interaction might be cell-type-specific, between the cd expressing cd8+ t cells and cd expressing cd4+ t cells. upon activation, killer cell lectin-like receptors showed mainly decreased expression. first discovered in nk cells, the expression of inhibitory receptors, and activating receptor klrc has also been reported in activated cd8+ t cells, involved in tcr signalling, but not in activated cd4+ t cells or naive t cells. the shared downregulation of these klrs along with that of some less-well-studied members suggests that their function might not be limited to effector cd8+ t cells, and that they are implicated more broadly in t-cell activation. surprisingly, members of both mhc classes i and ii were expressed in resting t cells. several genes encoding mhc class ii members showed decreased expression upon stimulation, while some mhc class i molecules were downregulated at 48– hours. granzymes secretion of cytotoxic granules is one of the major effector functions of cytotoxic t cells to induce apoptosis in target cells. perforins, granulysin and granzymes are the core components of the dense cytotoxic granules responsible for target cell lysis. most of the granzyme genes, but not gnly and prf were significantly regulated. gzma and gzmk were considerably downregulated throughout the experiments. it is likely that t cells have not acquired the full cytotoxic effector functions at this stage of the activation. however, gzmb was transcriptionally upregulated in both cd4+ and cd8+ t cells, and this was supported by data from a protein abundance assay. discussion genome-scale transcriptional profiling can add significant new information for better understanding t-cell activation as an important biological process of the immune response. previous efforts had examined t-cell activation at one single time point or addressed costimulatory signal effects using only a single experiment. using multiple donors, in this study we focused on the temporal, donor-independent gene expression patterns not only in the cd3+ t cells, but also in the cd4+ and cd8+ subsets. we identified donor-independent significantly regulated genes in t-cell activation in cd3+ t cells, and the cd4+ and cd8+ subsets. cd3+ t cells had far more significantly differentially expressed genes than cd4+ or cd8+ t cells. regardless, the transcription profiles of the pooled significant genes in t-cell activation shared largely similar patterns among the three populations. anti-cd3/anti-cd stimulation induced expression pattern changes of'immune response' genes which are consistent with the important roles of t cells in the adaptive immune response. not only well-known cytokines ) and cytokine receptors, cd icos), but also numerous novel ones, for t-cell activation, were differentially expressed. among the novel cytokines, the strongly upregulated chemokines might have important roles in enhancing t-cell activation in addition to their functions in cell trafficking, while the downregulated ccl il might rather be involved in maintaining the homeostasis of resting t cells. a number of cell-surface receptors not previously associated with t-cell activation, were differentially expressed, including downregulated ccr tnfrsf tnfrsf1a, ccr and il6r, and upregulated cd and cd a number of the receptors involved in tcr activation along with lck, zap and tyrobp, were all simultaneously downregulated. little is known about the expression and functions of klrs in resting t cells. the downregulated klr receptors are likely involved in the homeostasis of resting t cells. it is also possible that they regulate t-cell activation through tcr signalling. surprisingly, both mhc class i and class ii molecules were transcriptionally expressed in resting t cells, and mhc class ii molecules were significantly downregulated upon t-cell activation. mhc class i and class ii molecules are receptors on apcs, but not t cells, for the activation of cd8+ and cd4+ t cells, respectively. comparison of the expression patterns among the three populations provided further insights. different expression patterns between cd4+ and cd8+ t cells were observed, suggesting their subset specific involvement/functions in t-cell activation. among these genes, very little is known about xcl and slamf in t cells. genes with similar expression patterns between cd4+ and cd8+ t cells, but different than those in cd3+ t cells are likely involved in the communication between cd4+ and cd8+ subsets. these and the large number of novel genes in the context of t-cell activation that were identified in this study offer new research targets for a more complete understanding of t-cell activation.  our study captured novel temporal patterns of previously known but many novel, in the context of t-cell activation, genes ontologically classified under the term'immune response'. these patterns were reproducibly and robustly identified as donor independent, and were selectively confirmed by q-rt-pcr and protein-level assays. comprehensively integrating previous knowledge, we identified novel significant genes associated with the immune response in t cells, as well as subset specific genes, and genes implicated in the communication between cd4+ and cd8+ t cells. this study improves our understanding of the biology and the underlying regulation of t-cell activation in the natural cd3+ population, as well as in the cd4+ and cd8+ subsets. 
comparison of the microarray-based gene expression patterns between cd3+ t cells, and the cd4+ and cd8+ subsets revealed largely conserved, but not identical, transcriptional patterns. several receptors involved in tcr activation along with lck, zap and tyrobp were synchronously downregulated. significantly, a comparative genome-scale transcriptional analysis of two t-cell subsets against each other and against the naturally mixed population remains unexplored. chemokines induced during t-cell activation, but not previously identified in t cells, include ccl cxcl - -, and xcl. significantly, it made possible to identify the temporal patterns of many previously known t-cell activation genes, and also identify genes implicated in effector functions of and communication between cd4+ and cd8+ t cells. members of cell-surface receptors, none previously identified in the context of t-cell activation, were also downregulated. t-cell activation is an essential step of the immune response and relies on the tightly controlled orchestration of hundreds of genes/proteins, yet the cellular and molecular events underlying this complex process are not fully understood, especially at the genome-scale. we employed a gene-ontology-driven transcriptional analysis coupled with protein abundance assays in order to identify novel t-cell activation genes and cell-type-specific genes associated with the immune response. increased expression of other unexpected cytokines suggests their involvement in t-cell activation with their functions yet to be examined. differential expression of many receptors, not previously reported in the context of t-cell activation, including ccr ccr il1r il1rap, il6r, tnfrsf and tnfrsf1a, suggests their role in this immune process. this comparative genome-scale, transcriptional analysis of t-cell activation in the cd4+ and cd8+ subsets and the mixed cd3+ populations made possible the identification of many immune-response genes not previously identified in the context of t-cell activation.
 t-cell activation is an essential step of the immune response and relies on the tightly controlled orchestration of hundreds of genes/proteins, yet the cellular and molecular events underlying this complex process are not fully understood, especially at the genome-scale. significantly, a comparative genome-scale transcriptional analysis of two t-cell subsets against each other and against the naturally mixed population remains unexplored.  comparison of the microarray-based gene expression patterns between cd3+ t cells, and the cd4+ and cd8+ subsets revealed largely conserved, but not identical, transcriptional patterns. we employed a gene-ontology-driven transcriptional analysis coupled with protein abundance assays in order to identify novel t-cell activation genes and cell-type-specific genes associated with the immune response. we identified potential genes involved in the communication between the two subsets and would-be effector-function-specific genes. chemokines induced during t-cell activation, but not previously identified in t cells, include ccl cxcl - -, and xcl. increased expression of other unexpected cytokines suggests their involvement in t-cell activation with their functions yet to be examined. differential expression of many receptors, not previously reported in the context of t-cell activation, including ccr ccr il1r il1rap, il6r, tnfrsf and tnfrsf1a, suggests their role in this immune process. several receptors involved in tcr activation along with lck, zap and tyrobp were synchronously downregulated. members of cell-surface receptors, none previously identified in the context of t-cell activation, were also downregulated.  this comparative genome-scale, transcriptional analysis of t-cell activation in the cd4+ and cd8+ subsets and the mixed cd3+ populations made possible the identification of many immune-response genes not previously identified in the context of t-cell activation. significantly, it made possible to identify the temporal patterns of many previously known t-cell activation genes, and also identify genes implicated in effector functions of and communication between cd4+ and cd8+ t cells. 