BACKGROUND
acquired aplastic anemia  is a rare bone marrow failure state characterized by marrow hypocellularity and low peripheral blood cell counts  <cit> .
although the exact pathogenic mechanism is still unknown, beside a primary stem cell defect  <cit>  and a disturbed microenvironment  <cit> , pre-/clinical data suggest an immune-mediated destruction of marrow progenitor/stem cells  <cit> .
similar to other autoimmune diseases, antigen-specific t cells could be expanded from the bone marrow of aa patients and likely mediate organ-specific cytotoxicity to hematopoietic stem cells and progenitor cells  <cit> .
in addition, it has been reported that the bone marrow of aa patients contains increased numbers of apoptotic cells  <cit>  and that hematopoietic stem cells of aa patients express fas antigen  <cit> .
peripheral blood and bone marrow t-cells show signs of activation  <cit> , secrete high levels of interferon-gamma  and tumor-necrosis factor alpha .
these two cytokines may suppress the proliferation of early and late hematopoietic progenitor cells and initiate apoptosis by induction of fas on cd34+ stem cells  <cit> .
most recently, some groups reported a polarization of cd4+ t-cells towards a type- <dig> immune response that leads to activation of cytotoxic cd8+ t-cells with destruction of marrow stem cells  <cit> .
however, genes important for the recruitment of effector t-cells to the bone marrow as target organ of autoimmunity and regulatory molecules directing their differentiation have not been identified so far.
therefore, we performed a microarray study comparing gene expression profiles of circulating and marrow-derived t-cells at initial presentation and after hematological recovery in order to elucidate more details of the mechanism resulting in severe bone marrow failure.
RESULTS
microarray analysis
comparative gene expression profiling was performed in severe aplastic anemia  patients and healthy controls by analyzing pooled cd3+ t-cells isolated from peripheral blood  and bone marrow  samples .
each oligonucleotide microarray  contained  <dig>  human probesets and in our experiments discovered between  <dig> % and  <dig> % "present calls" as calculated by the statistical detection algorithm of affymetrix.
in general, "present calls" of approximately 50% provide confidential results.
scaled factors which were used to normalize the arrays to an average intensity ranged from  <dig>  to  <dig>  showing the high reproducibility of the experimental procedure.
furthermore, the hybridisation was monitored by the usage of hybridisation controls comparing total rna amount and final signal intensity on the array .
taken together, our data reached a highly acceptable quality level for further analysis.
to eliminate genes which may be individually regulated independent of disease we focused in our study only on genes coregulated in both of the analyzed saa pools  in comparison to the normal controls , each separately for pb and bm samples.
the potentially disease-specific regulation comprised a wide variety of genes belonging to different functional classes, such as immune response, proliferation/cell growth, biosynthesis/metabolism, signal transduction and apoptosis .
the most interesting genes are summarized below, whereas the entire data set is available at the geo database  <cit>  under the accession number gse <dig>
gene expression profiles of t-cells from saa patients at initial diagnosis
the circulating t-cells isolated from the pb samples of saa patients exhibited a broadly normal gene expression profile with dysregulation of only  <dig> genes  in both of the analyzed saa pools.
in contrast, in comparison to the control pool t-cells isolated from bm aspirations showed a dysregulation of  <dig> genes , which were coregulated in both saa pools.
among the regulated genes identified in circulating t-cells some were also differentially expressed in bm-derived t-lymphocytes  and other molecules were only regulated in pb samples .
differentially expressed genes, which were exclusively found in bm-infiltrating t-cells, were classified into several functional categories .
these genes included molecules involved in immune responses and their regulation with the following fold changes for saa pool i and iia, respectively: pf- <dig> , cd <dig> , ncf- <dig> , ccr <dig> , cx3cr <dig>  and other chemokine receptors and ligands.
many cytokines/growth factors and their receptors, which play an important role in inflammation and endogenous response to infections, were found to be dysregulated like il-1α , irak <dig> , ltβ , toll-like receptor  <dig>  and cias <dig> .
furthermore molecules important for the regulation of proliferation and cell cycling were dysregulated .
finally, several transcription factors were found to be differentially expressed in saa patients .
to obtain more information, we also set a minimum fold change of  <dig>  revealing an upregulation of genes in bm-derived cd3+ t-cells from saa patients playing an important role in the regulation of apoptosis, like tradd , traf <dig> , perforin- <dig> , granzyme h  and granzyme b .
in order to examine whether the obtained differential gene expression profiles in the analyzed saa patients simply reflect an over- or under-representation of t-cell subpopulations, the differential gene expression pattern identified in circulating t-cells of saa patients  were compared with gene expression profiles of effector memory versus naive cd4+ and cd8+ t-cell populations generated from normal donors  <cit> .
the comparative analysis of effector memory and naïve cd4+  shows that  <dig> genes  are statistically singificantly regulated in one of the analyzed subpopulations .
interestingly, only  <dig>  of these genes exhibited a similar expression pattern in the circulating cd3+ t-cells of the saa patient pools .
gene expression profiles of cd3+ t-cells from aa patients in remission
gene expression profiles  were performed of cd3+ t-cells isolated from pb and bm samples of  <dig> saa patients after achieving a hematological remission following immunosuppressive therapy .
data were analyzed with statistical tools as described in the method part and directly compared to their respective gene expression profile at initial presentation .
in order to provide a better overview, we performed a hierarchical cluster analysis including genes that tend to normalize with respect to the gene expression levels detected in the normal control pools .
in total, we identified  <dig> of  <dig> regulated genes in the bm samples of the analyzed saa patients turning to nearly normal gene expression levels after successful immunotherapy.
this category mainly included genes involved in the regulation of immune response like ncf <dig>  in the pb samples of the saa pool only one of  <dig> regulated genes  tended to expression levels close to the normal control pool, but its function in the immune system is unknown.
furthermore, the comparison of the gene expression profiles  revealed a group of several genes with a very high difference  in the expression level before and after successful immunotherapy .
most of these genes are important during inflammation and regulation of immune response, such as platelet-factor  <dig> , proteoglycan  <dig> und  <dig> , il-1α, irak <dig>  osteopontin , receptor for fc fragment of ige  or rnase <dig>  the fold changes of regulated candidate genes are summarized in table  <dig>
validation of microarray data by real-time rt-pcr
to confirm the microarray results, quantitative real-time rt-pcr of selected candidate genes was performed using the original patient sample pools  and patient  <dig> in hematological remission.
because of limited amounts of pooled rna,  <dig> genes were selected for quantification of gene expression using a new pool of normal bm samples.
even with this new sample pool the regulation of gene expression could be confirmed for the  <dig> analyzed genes .
for example, separate microarray experiments revealed an increased expression for cd <dig> in pools i and iia , which could be confirmed by real-time rt-pcr .
in general, real-time rt-pcr results correlated well with the differential gene expression data of the microarray experiments.
discussion
in acquired aplastic anemia, the evidence of an autoimmune pathogenesis is mostly indirect and the characterization of the underlying immune response is incomplete mainly due to technical difficulties resulting from the disease-specific hypocellularity.
however, there is growing evidence that aa results from autoaggressive destruction of hematopoietic stem cells and progenitors mediated by t-cells recognizing inciting target antigens  <cit> .
several groups have identified clonal t-cell expansion  <cit> , proinflammatory cytokine production  <cit>  and t-cell mediated cytotoxicity to cd34+ stem cells  <cit>  supporting an antigen-driven t-cell response.
most recently, we could characterize the antigen-binding sites of clonally expanded t-cells in saa patients and identified the emergence of new t-cell clones after successful immunosuppression  <cit> , suggesting a regulatory t-cell population contributing to the recovery of hematopoiesis.
in this study, we applied microarray technology to characterize the immune response of the t-cell system in saa.
the design of our experiments differed from most recently reported transcript profiles of aa patients  <cit>  in the following important issues:  the untreated patients included in this study were diagnosed as saa with similar clinical characteristics, hla background, response to therapy and without an expanded pnh clone.
gene profiles of cd3+ t-cells from pb and bm samples of the same saa patients and healthy controls were compared, as the pathological t-cell response is expected in the bone marrow as target organ of aa.
furthermore, the gene profiles of saa patients were comparatively analyzed with respect to normal gene expression before and after successful immunotherapy in order to identify candidate genes relevant for the recovery of hematopoiesis.
as expected the gene profiles of cd3+ t-cells isolated from pb samples differed significantly from the expression pattern found in bm-derived t-cells of saa patients.
the number of differentially expressed genes in circulating t-cells of saa patients compared to healthy controls was surprisingly low: only  <dig> genes were differentially expressed in peripheral t-cells of the patients pools.
interestingly among these genes, a negative regulator of cytokine response, the zinc finger protein tnfaip <dig>  was upregulated.
the induction of tnfaip <dig> may result from cellular counterregulation in response to high serum levels of tnf and il- <dig>  <cit> , as this molecule specifically inhibits the respective signal transduction pathways  <cit> .
two important multifunctional regulators of hematopoiesis, mip1α  <cit>  and skil  <cit> , were regulated in both peripheral and marrow-derived t-cells at initial diagnosis.
whereas mip1α exerts potentially suppressive effects on the early myelopoiesis in aa  <cit> , skil functions as negative regulator of tgfβ signaling  <cit>  and its induction may also represent a counterregulatory event.
in comparison to normal controls, marrow-derived cd3+ t-cells showed a large scale of differentially expressed genes indicating that t-cell dysregulation in the bm as target organ is a critical part of the pathological immune response.
the regulation of  <dig> genes also demonstrates that the bone marrow failure results from a rather complex genetic program involving chemokines, cytokines, growth factors, and their receptors.
we could identify the induction of several molecules playing key roles in the regulation of th <dig> immune responses, such as ccr <dig> and cx3cr <dig>  <cit> , which are also important in other autoimmune diseases such as multiple sclerosis  <cit> , and cd <dig>  a surface-bound ectopeptidase expressed at high levels on th <dig> differentiated t-cells  <cit> .
the microarray study identified several genes like cd <dig>  pf <dig> and ncf <dig> with distinct immunregulatory functions.
as these candidate genes were responding to successful immunotherapy, they might play a key role in the immunpathogenesis of aa and recovery of hematopoiesis after immunosuppression: specific blocking of cd <dig>  which was upregulated at initial diagnosis and normalized in hematological remission, suppresses the proliferation of autoreactive t-cells with upregulation of tgf-β <dig> and downregulation of tnf-α  <cit> ; furthermore cd <dig> favors cytotoxic effector functions in cd8+ t-cells  <cit> .
specific binding of the chemokine pf <dig>  which was initially enourmosly downregulated and almost normalized after immunotherapy, prevents the release of il- <dig> and ifn-γ in activated t-cells and may especially suppress autoreactive t cells in the presence of proinflammatory cytokines  <cit> .
ncf <dig>  a gene important for oxidative burst formation, regulates the severity of other autoimmune diseases and modulates directly the level of t-cell dependent autoimmune responses  <cit> .
taken together, these candidate genes might be highly relevant for the autoreactive t-cell attack in aa and the induction of peripheral tolerance with recovery of the hematopoiesis following immunotherapy.
in contrast to zeng et al.
<cit>  we could not identify an upregulation of several other chemokines/-receptors , which play a role in other autoimmune diseases.
also inconsistent with already reported transcript profiles  <cit>  we identifed a downregulation of several molecules involved in innate immune responses, such as toll-like receptor  <dig>  il- <dig> receptor-associated kinase , il- <dig> and its receptor in our saa patient pools.
the dysregulation of innate immunity in saa patients may result in increased proinflammatory cytokine production towards bacterial challenge as observed in irak3-/- mice  <cit> .
in accordance to earlier reports  <cit>  we could identify a downregulation of il-1α/β in bm-derived t-cells at initial presentation with normalization of the expression level for il-1α after hematological recovery.
interestingly, the administration of il- <dig> accelerates hematopoietic reconstitution after chemotherapy induced myelosuppression in vivo  <cit>  underlining its potential role in marrow failure syndromes.
additionally, the gene expression level of an important regulator of nfκb-dependent proinflammatory signals, cias <dig>  was regulated enormously in the pre- and post-therapeutic saa pools.
interestingly, mutations of cias <dig> cause several autoimmune inflammatory syndromes  <cit>  and its downregulation in saa patients may contribute to the immunpathology of aa by augmentation of proinflammatory signals.
as weakly regulated molecules may also have important influence on disease development, we additionally analyzed the gene expression profiles for a minimum fold change of  <dig>  and could reveal an upregulation of several genes related to cell-mediated cytotoxicity and apoptosis, such as perforin- <dig>  granzyme h and b  <cit> .
the involvement of cytotoxic granules in the induction of target cell apoptosis has already been shown in aa  <cit>  and other autoimmune disorders  <cit> .
interestingly, traf and tradd, two mediators of the tnfr familiy for cell activation, survival and antiapoptotic function  <cit>  were upregulated in bm-derived t-cells and may be protective for the effector population in the presence of high tnfα levels.
CONCLUSIONS
in summary, our microarray study revealed a th1/tc <dig> phenotype in bone marrow-derived t-cells of saa patients at initial presentation.
whereas the potential mechanism for the recruitment of effector t-cell to the bone marrow remained unclear, we could identify several regulators highly relevant for the immunpathogenesis of aa and likely contributing to the recovery of hematopoiesis.
especially the identified regulators of proinflammatory cytokine signaling  and molecules with distinct immunregulatory properties in controlling autoreactive t-cells  may represent potential targets for the design of new therapeutic strategies.

 acquired aplastic anemia is a rare bone marrow failure state characterized by marrow hypocellularity and low peripheral blood cell counts. although the exact pathogenic mechanism is still unknown, beside a primary stem cell defect and a disturbed microenvironment, pre-/clinical data suggest an immune-mediated destruction of marrow progenitor/stem cells. similar to other autoimmune diseases, antigen-specific t cells could be expanded from the bone marrow of aa patients and likely mediate organ-specific cytotoxicity to hematopoietic stem cells and progenitor cells. in addition, it has been reported that the bone marrow of aa patients contains increased numbers of apoptotic cells and that hematopoietic stem cells of aa patients express fas antigen. peripheral blood and bone marrow t-cells show signs of activation, secrete high levels of interferon-gamma and tumor-necrosis factor alpha. these two cytokines may suppress the proliferation of early and late hematopoietic progenitor cells and initiate apoptosis by induction of fas on cd34+ stem cells. most recently, some groups reported a polarization of cd4+ t-cells towards a type- immune response that leads to activation of cytotoxic cd8+ t-cells with destruction of marrow stem cells. however, genes important for the recruitment of effector t-cells to the bone marrow as target organ of autoimmunity and regulatory molecules directing their differentiation have not been identified so far. therefore, we performed a microarray study comparing gene expression profiles of circulating and marrow-derived t-cells at initial presentation and after hematological recovery in order to elucidate more details of the mechanism resulting in severe bone marrow failure.  microarray analysis comparative gene expression profiling was performed in severe aplastic anemia patients and healthy controls by analyzing pooled cd3+ t-cells isolated from peripheral blood and bone marrow samples. each oligonucleotide microarray contained human probesets and in our experiments discovered between % and % "present calls" as calculated by the statistical detection algorithm of affymetrix. in general, "present calls" of approximately 50% provide confidential results. scaled factors which were used to normalize the arrays to an average intensity ranged from to showing the high reproducibility of the experimental procedure. furthermore, the hybridisation was monitored by the usage of hybridisation controls comparing total rna amount and final signal intensity on the array. taken together, our data reached a highly acceptable quality level for further analysis. to eliminate genes which may be individually regulated independent of disease we focused in our study only on genes coregulated in both of the analyzed saa pools in comparison to the normal controls, each separately for pb and bm samples. the potentially disease-specific regulation comprised a wide variety of genes belonging to different functional classes, such as immune response, proliferation/cell growth, biosynthesis/metabolism, signal transduction and apoptosis. the most interesting genes are summarized below, whereas the entire data set is available at the geo database under the accession number gse gene expression profiles of t-cells from saa patients at initial diagnosis the circulating t-cells isolated from the pb samples of saa patients exhibited a broadly normal gene expression profile with dysregulation of only genes in both of the analyzed saa pools. in contrast, in comparison to the control pool t-cells isolated from bm aspirations showed a dysregulation of genes, which were coregulated in both saa pools. among the regulated genes identified in circulating t-cells some were also differentially expressed in bm-derived t-lymphocytes and other molecules were only regulated in pb samples. differentially expressed genes, which were exclusively found in bm-infiltrating t-cells, were classified into several functional categories. these genes included molecules involved in immune responses and their regulation with the following fold changes for saa pool i and iia, respectively: pf-, cd, ncf-, ccr, cx3cr and other chemokine receptors and ligands. many cytokines/growth factors and their receptors, which play an important role in inflammation and endogenous response to infections, were found to be dysregulated like il-1α, irak, ltβ, toll-like receptor and cias. furthermore molecules important for the regulation of proliferation and cell cycling were dysregulated. finally, several transcription factors were found to be differentially expressed in saa patients. to obtain more information, we also set a minimum fold change of revealing an upregulation of genes in bm-derived cd3+ t-cells from saa patients playing an important role in the regulation of apoptosis, like tradd, traf, perforin-, granzyme h and granzyme b. in order to examine whether the obtained differential gene expression profiles in the analyzed saa patients simply reflect an over- or under-representation of t-cell subpopulations, the differential gene expression pattern identified in circulating t-cells of saa patients were compared with gene expression profiles of effector memory versus naive cd4+ and cd8+ t-cell populations generated from normal donors. the comparative analysis of effector memory and naïve cd4+ shows that genes are statistically singificantly regulated in one of the analyzed subpopulations. interestingly, only of these genes exhibited a similar expression pattern in the circulating cd3+ t-cells of the saa patient pools. gene expression profiles of cd3+ t-cells from aa patients in remission gene expression profiles were performed of cd3+ t-cells isolated from pb and bm samples of saa patients after achieving a hematological remission following immunosuppressive therapy. data were analyzed with statistical tools as described in the method part and directly compared to their respective gene expression profile at initial presentation. in order to provide a better overview, we performed a hierarchical cluster analysis including genes that tend to normalize with respect to the gene expression levels detected in the normal control pools. in total, we identified of regulated genes in the bm samples of the analyzed saa patients turning to nearly normal gene expression levels after successful immunotherapy. this category mainly included genes involved in the regulation of immune response like ncf in the pb samples of the saa pool only one of regulated genes tended to expression levels close to the normal control pool, but its function in the immune system is unknown. furthermore, the comparison of the gene expression profiles revealed a group of several genes with a very high difference in the expression level before and after successful immunotherapy. most of these genes are important during inflammation and regulation of immune response, such as platelet-factor, proteoglycan und, il-1α, irak osteopontin, receptor for fc fragment of ige or rnase the fold changes of regulated candidate genes are summarized in table validation of microarray data by real-time rt-pcr to confirm the microarray results, quantitative real-time rt-pcr of selected candidate genes was performed using the original patient sample pools and patient in hematological remission. because of limited amounts of pooled rna, genes were selected for quantification of gene expression using a new pool of normal bm samples. even with this new sample pool the regulation of gene expression could be confirmed for the analyzed genes. for example, separate microarray experiments revealed an increased expression for cd in pools i and iia, which could be confirmed by real-time rt-pcr. in general, real-time rt-pcr results correlated well with the differential gene expression data of the microarray experiments. discussion in acquired aplastic anemia, the evidence of an autoimmune pathogenesis is mostly indirect and the characterization of the underlying immune response is incomplete mainly due to technical difficulties resulting from the disease-specific hypocellularity. however, there is growing evidence that aa results from autoaggressive destruction of hematopoietic stem cells and progenitors mediated by t-cells recognizing inciting target antigens. several groups have identified clonal t-cell expansion, proinflammatory cytokine production and t-cell mediated cytotoxicity to cd34+ stem cells supporting an antigen-driven t-cell response. most recently, we could characterize the antigen-binding sites of clonally expanded t-cells in saa patients and identified the emergence of new t-cell clones after successful immunosuppression, suggesting a regulatory t-cell population contributing to the recovery of hematopoiesis. in this study, we applied microarray technology to characterize the immune response of the t-cell system in saa. the design of our experiments differed from most recently reported transcript profiles of aa patients in the following important issues: the untreated patients included in this study were diagnosed as saa with similar clinical characteristics, hla background, response to therapy and without an expanded pnh clone. gene profiles of cd3+ t-cells from pb and bm samples of the same saa patients and healthy controls were compared, as the pathological t-cell response is expected in the bone marrow as target organ of aa. furthermore, the gene profiles of saa patients were comparatively analyzed with respect to normal gene expression before and after successful immunotherapy in order to identify candidate genes relevant for the recovery of hematopoiesis. as expected the gene profiles of cd3+ t-cells isolated from pb samples differed significantly from the expression pattern found in bm-derived t-cells of saa patients. the number of differentially expressed genes in circulating t-cells of saa patients compared to healthy controls was surprisingly low: only genes were differentially expressed in peripheral t-cells of the patients pools. interestingly among these genes, a negative regulator of cytokine response, the zinc finger protein tnfaip was upregulated. the induction of tnfaip may result from cellular counterregulation in response to high serum levels of tnf and il-, as this molecule specifically inhibits the respective signal transduction pathways. two important multifunctional regulators of hematopoiesis, mip1α and skil, were regulated in both peripheral and marrow-derived t-cells at initial diagnosis. whereas mip1α exerts potentially suppressive effects on the early myelopoiesis in aa, skil functions as negative regulator of tgfβ signaling and its induction may also represent a counterregulatory event. in comparison to normal controls, marrow-derived cd3+ t-cells showed a large scale of differentially expressed genes indicating that t-cell dysregulation in the bm as target organ is a critical part of the pathological immune response. the regulation of genes also demonstrates that the bone marrow failure results from a rather complex genetic program involving chemokines, cytokines, growth factors, and their receptors. we could identify the induction of several molecules playing key roles in the regulation of th immune responses, such as ccr and cx3cr, which are also important in other autoimmune diseases such as multiple sclerosis, and cd a surface-bound ectopeptidase expressed at high levels on th differentiated t-cells. the microarray study identified several genes like cd pf and ncf with distinct immunregulatory functions. as these candidate genes were responding to successful immunotherapy, they might play a key role in the immunpathogenesis of aa and recovery of hematopoiesis after immunosuppression: specific blocking of cd which was upregulated at initial diagnosis and normalized in hematological remission, suppresses the proliferation of autoreactive t-cells with upregulation of tgf-β and downregulation of tnf-α; furthermore cd favors cytotoxic effector functions in cd8+ t-cells. specific binding of the chemokine pf which was initially enourmosly downregulated and almost normalized after immunotherapy, prevents the release of il- and ifn-γ in activated t-cells and may especially suppress autoreactive t cells in the presence of proinflammatory cytokines. ncf a gene important for oxidative burst formation, regulates the severity of other autoimmune diseases and modulates directly the level of t-cell dependent autoimmune responses. taken together, these candidate genes might be highly relevant for the autoreactive t-cell attack in aa and the induction of peripheral tolerance with recovery of the hematopoiesis following immunotherapy. in contrast to zeng et al. we could not identify an upregulation of several other chemokines/-receptors, which play a role in other autoimmune diseases. also inconsistent with already reported transcript profiles we identifed a downregulation of several molecules involved in innate immune responses, such as toll-like receptor il- receptor-associated kinase, il- and its receptor in our saa patient pools. the dysregulation of innate immunity in saa patients may result in increased proinflammatory cytokine production towards bacterial challenge as observed in irak3-/- mice. in accordance to earlier reports we could identify a downregulation of il-1α/β in bm-derived t-cells at initial presentation with normalization of the expression level for il-1α after hematological recovery. interestingly, the administration of il- accelerates hematopoietic reconstitution after chemotherapy induced myelosuppression in vivo underlining its potential role in marrow failure syndromes. additionally, the gene expression level of an important regulator of nfκb-dependent proinflammatory signals, cias was regulated enormously in the pre- and post-therapeutic saa pools. interestingly, mutations of cias cause several autoimmune inflammatory syndromes and its downregulation in saa patients may contribute to the immunpathology of aa by augmentation of proinflammatory signals. as weakly regulated molecules may also have important influence on disease development, we additionally analyzed the gene expression profiles for a minimum fold change of and could reveal an upregulation of several genes related to cell-mediated cytotoxicity and apoptosis, such as perforin- granzyme h and b. the involvement of cytotoxic granules in the induction of target cell apoptosis has already been shown in aa and other autoimmune disorders. interestingly, traf and tradd, two mediators of the tnfr familiy for cell activation, survival and antiapoptotic function were upregulated in bm-derived t-cells and may be protective for the effector population in the presence of high tnfα levels.  in summary, our microarray study revealed a th1/tc phenotype in bone marrow-derived t-cells of saa patients at initial presentation. whereas the potential mechanism for the recruitment of effector t-cell to the bone marrow remained unclear, we could identify several regulators highly relevant for the immunpathogenesis of aa and likely contributing to the recovery of hematopoiesis. especially the identified regulators of proinflammatory cytokine signaling and molecules with distinct immunregulatory properties in controlling autoreactive t-cells may represent potential targets for the design of new therapeutic strategies. 
to overcome this technical problem we performed a microarray analysis of cd3+ t-cells derived from bone marrow aspirates and peripheral blood samples of newly diagnosed aa patients and healthy volunteers. two aa patients were additionally analyzed after achieving a partial remission following immunosuppression. among more than transcripts, genes were differentially expressed in the bone marrow of aa patients compared to healthy controls. our study suggests a pivotal role of th1/tc differentiated t-cells in immune-mediated marrow destruction of aa patients. in hematological remission the expression levels of several candidate genes tend to normalize, such as immune regulators and genes involved in proinflammatory immune response. most importantly, immune regulatory genes could be identified, which are likely involved in the recovery of hematopoiesis and may help to design new therapeutic strategies in bone marrow failure syndromes. the regulation of selected candidate genes was confirmed by real-time rt-pcr. aplastic anemia is a bone marrow failure syndrome mostly characterized by an immune-mediated destruction of marrow hematopoietic progenitor/stem cells. the resulting hypocellularity limits a detailed analysis of the cellular immune response. dysregulated genes are involved in t-cell mediated cytotoxicity, immune response of th differentiated t-cells, and major regulators of immune function.
 aplastic anemia is a bone marrow failure syndrome mostly characterized by an immune-mediated destruction of marrow hematopoietic progenitor/stem cells. the resulting hypocellularity limits a detailed analysis of the cellular immune response. to overcome this technical problem we performed a microarray analysis of cd3+ t-cells derived from bone marrow aspirates and peripheral blood samples of newly diagnosed aa patients and healthy volunteers. two aa patients were additionally analyzed after achieving a partial remission following immunosuppression. the regulation of selected candidate genes was confirmed by real-time rt-pcr.  among more than transcripts, genes were differentially expressed in the bone marrow of aa patients compared to healthy controls. dysregulated genes are involved in t-cell mediated cytotoxicity, immune response of th differentiated t-cells, and major regulators of immune function. in hematological remission the expression levels of several candidate genes tend to normalize, such as immune regulators and genes involved in proinflammatory immune response.  our study suggests a pivotal role of th1/tc differentiated t-cells in immune-mediated marrow destruction of aa patients. most importantly, immune regulatory genes could be identified, which are likely involved in the recovery of hematopoiesis and may help to design new therapeutic strategies in bone marrow failure syndromes. 