[show abstract][hide abstract] ABSTRACT: Hematopoietic effects of interferon-gamma (IFN-gamma) may be responsible for certain aspects of the pathology seen in bone marrow failure syndromes, including aplastic anemia (AA), paroxysmal nocturnal hemoglobinuria (PNH), and some forms of myelodysplasia (MDS). Overexpression of and hematopoietic inhibition by IFN-gamma has been observed in all of these conditions. In vitro, IFN-gamma exhibits strong inhibitory effects on hematopoietic progenitor and stem cells. Previously, we have studied the transcriptome of CD34 cells derived from patients with bone marrow failure syndromes and identified characteristic molecular signatures common to some of these conditions. In this report, we have investigated genome-wide expression patterns after exposure of CD34 and bone marrow stroma cells derived from normal bone marrow to IFN-gamma in vitro and have detected profound changes in the transcription profile. Some of these changes were concordant in both stroma and CD34 cells, whereas others were specific to CD34 cells. In general, our results were in agreement with the previously described function of IFN-gamma in CD34 cells involving activation of apoptotic pathways and immune response genes. Comparison between the IFN-gamma transcriptome in normal CD34 cells and changes previously detected in CD34 cells from AA and PNH patients reveals the presence of many similarities that may reflect molecular signature of in vivo IFN-gamma exposure.
[show abstract][hide abstract] ABSTRACT: Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired stem cell disorder characterized clinically by intravascular hemolysis, venous thrombosis, and bone marrow failure. Despite elucidation of the biochemical and molecular defects in PNH, the pathophysiology of clonal expansion of glycosylphosphatidylinositol-anchored protein (GPI-AP)-deficient cells remains unexplained. In pursuit of evidence of differences between GPI-AP-normal and -deficient CD34 cells, we determined gene expression profiles of isolated marrow CD34 cells of each phenotype from PNH patients and healthy donors, using DNA microarrays. Pooled and individual patient samples revealed consistent gene expression patterns relative to normal controls. GPI-AP-normal cells from PNH patients showed upregulation of genes involved in apoptosis and the immune response. Conversely, genes associated with antiapoptotic function and hematopoietic cell proliferation and differentiation were downregulated in these cells. In contrast, the PNH clone of GPI-AP-deficient cells appeared more similar to CD34 cells of healthy individuals. Gene chip data were confirmed by other methods. Similar gene expression patterns were present in PNH that was predominantly hemolytic as in PNH associated with aplastic anemia. Our results implicate an environmental influence on hematopoietic cell proliferation, in which the PNH clone evades immune attack and destruction, while normal cells suffer a stress response followed by programmed cell death.
[show abstract][hide abstract] ABSTRACT: Aneuploidy, especially monosomy 7 and trisomy 8, is a frequent cytogenetic abnormality in the myelodysplastic syndromes (MDSs). Patients with monosomy 7 and trisomy 8 have distinctly different clinical courses, responses to therapy, and survival probabilities. To determine disease-specific molecular characteristics, we analyzed the gene expression pattern in purified CD34 hematopoietic progenitor cells obtained from MDS patients with monosomy 7 and trisomy 8 using Affymetrix GeneChips. Two methods were employed: standard hybridization and a small-sample RNA amplification protocol for the limited amounts of RNA available from individual cases; results were comparable between these 2 techniques. Microarray data were confirmed by gene amplification and flow cytometry using individual patient samples. Genes related to hematopoietic progenitor cell proliferation and blood cell function were dysregulated in CD34 cells of both monosomy 7 and trisomy 8 MDS. In trisomy 8, up-regulated genes were primarily involved in immune and inflammatory responses, and down-regulated genes have been implicated in apoptosis inhibition. CD34 cells in monosomy 7 showed up-regulation of genes inducing leukemia transformation and tumorigenesis and apoptosis and down-regulation of genes controlling cell growth and differentiation. These results imply distinct molecular mechanisms for monosomy 7 and trisomy 8 MDS and implicate specific pathogenic pathways.
[show abstract][hide abstract] ABSTRACT: Immune-mediated destruction of hematopoietic stem and progenitor cells is pathophysiologic in most cases of aplastic anemia (AA). We have successfully determined the gene expression profile of the marrow CD34+ target cells in AA. T cells producing IFN-gamma and TNF-alpha have been implicated in hematopoietic destruction in AA. We sought to characterize T cells as immune mediators using the microarray approach.
We applied Affymetrix GeneChip techniques to determine the detailed profile of mRNA expression of CD4+ and CD8+ cells from the BM of newly diagnosed AA patients and healthy volunteers. For validation, we confirmed our microarray results using quantitative real-time PCR.
Compared to healthy controls, there were 178 and 183 differentially expressed genes in patients' CD4+ cells and CD8+ T cells, respectively; activities of 22 selected genes were confirmed using real-time PCR. Dysregulated genes included those encoding cytokines/chemokines, and involved in transcription regulation, calcium and ion channel formation, and cell adhesion. Unexpected findings were overexpression of toll-like receptor genes in marrow CD4+ cells of patients and of genes for killer-cell immunoglobulin-like receptors (KIR) in AA marrow CD8+ cells.
Our detailed results at the mRNA level provide insights into the mechanism of AA. Both innate and adaptive immune responses of CD4+ and CD8+ T cells appear to be active in immune-mediated marrow destruction. A variety of cytokines and chemokines active in pathophysiologic cells likely play important roles in the recruitment and activation of lymphocytes to cytotoxic effectors for marrow hematopoietic target cells in AA.
[show abstract][hide abstract] ABSTRACT: Paroxysmal nocturnal haemoglobinuria (PNH) results from acquired mutations in the PIG-A gene of an haematopoietic stem cell, leading to defective biosynthesis of glycosylphosphatidylinositol (GPI) anchors and deficient expression of GPI-anchored proteins on the surface of the cell's progeny. Some laboratory and clinical findings have suggested genomic instability to be intrinsic in PNH; this possibility has been supported by mutation analysis of hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene abnormalities. However, the HPRT assay examines lymphocytes in peripheral blood (PB), and T cells may be related to the pathophysiology of PNH. We analysed the molecular and functional features of HPRT mutants in PB mononuclear cells from eleven PNH patients. CD8 T cells predominated in these samples; approximately half of the CD8 cells lacked GPI-anchored protein expression, while only a small proportion of CD4 cells appeared to derive from the PNH clone. The HPRT mutant frequency (Mf) in T lymphocytes from PNH patients was significantly higher than in healthy controls. The majority of the mutant T lymphocyte clones were of CD4 phenotype, and they had phenotypically normal GPI-anchored protein expression. In PNH patients, the majority of HPRT mutant clones were contained within the Vbeta2 T cell receptor (TCR) subfamily, which was oligoclonal by complementarity-determining region three (CDR3) size analysis. Our results are more consistent with detection of uniform populations of expanded T cell clones, which presumably acquired HPRT mutations during antigen-driven cell proliferation, and not due to an increased Mf in PNH. HPRT mutant analysis does not support underlying genomic instability in PNH.
British Journal of Haematology 06/2004; 125(3):383-91. · 4.94 Impact Factor
[show abstract][hide abstract] ABSTRACT: An immune pathophysiology for acquired aplastic anemia (AA) has been inferred from the responsiveness of the patients to immunosuppressive therapies and experimental laboratory data. To address the transcriptome of hematopoietic cells in AA, we undertook GeneChip analysis of the extremely limited numbers of progenitor and stem cells in the marrow of patients with this disease. We pooled total RNA from highly enriched bone marrow CD34 cells of 36 patients with newly diagnosed AA and 12 healthy volunteers for analysis on oligonucleotide chips. A large number of genes implicated in apoptosis and cell death showed markedly increased expression in AA CD34 cells, and negative proliferation control genes also had increased activity. Conversely, cell cycle progress-enhancing genes showed low expression in AA. Cytokine/chemokine signal transducer genes, stress response genes, and defense/immune response genes were up-regulated, as anticipated from other evidence of the heightened immune activity in AA patients' marrow. In summary, detailed genetic analysis of small numbers of hematopoietic progenitor cells is feasible even in marrow failure states where such cells are present in very small numbers. The gene expression profile of primary human CD34 hematopoietic stem cells from AA was consistent with a stressed, dying, and immunologically activated target cell population. Many of the genes showing differential expression in AA deserve further detailed analysis, including comparison with other marrow failure states and autoimmune disease.
[show abstract][hide abstract] ABSTRACT: The efficacy of immunosuppressive therapy in aplastic anemia (AA) provides the strongest argument to support its immune-mediated pathophysiology. While several immunosuppressive effects of antithymocyte globulin (ATG) can be demonstrated in vitro, some reports have implied that the activity of ATG in AA may be rather due to a variety of positive hematopoietic effects. We studied the effects of horse (h) and rabbit (r) ATG on marrow progenitors in vitro. Both types of ATG bound to CD34 cells and, in colony assays performed with total marrow cells, hATG had a dose-dependent, triphasic effect, with maximal increase in colony formation between 1 and 10 microg/ml and inhibition between 100 and 1000 microg/ml. As determined using CD34 cells, these effects did not require accessory cells. rATG showed similar activity, but was about 10-fold more potent than hATG. In the presence of complement, no increased cytotoxicity was observed. At concentrations equivalent to those measured in patients immediately after infusion, ATG showed moderate suppression of colony formation, while the stimulatory concentrations in vitro correspond to those seen in vivo within the first weeks after ATG administration. In control experiments, the patterns of the biologic effects of preimmune rIgG or hIgG preparations were similar to those of rATG and hATG, indicating a nonspecific nature of the effects of ATG on progenitor cells. Biological activity in methylcellulose cultures was observed with the F(ab)(2) fragments but was not found in purified Fc IgG. In summary, the spectrum of effects of ATG on hematopoietic progenitors is dependent upon the concentrations of ATG and may not be related to its antigenic specificity.
The Hematology Journal 02/2004; 5(3):255-61. · 1.86 Impact Factor
[show abstract][hide abstract] ABSTRACT: Aplastic anaemia is a bone-marrow-failure syndrome characterised by low blood-cell counts and fatty bone marrow. In most cases, no obvious aetiological factor can be identified. However, clinical responses to immunosuppression strongly suggest an immune pathophysiology.
To test the hypothesis that aplastic anaemia results from antigen-specific lymphocyte attack against haemopoietic tissue, we analysed effector immunity, seeking especially dominant specific T-cell responses. Blood samples from 54 patients with aplastic anaemia were subjected to flow cytometry to define T-cell-receptor Vbeta-chain usage and expansion of particular Vbeta subsets. We measured the size distribution of the complementarity-determining region 3 (CDR3) for expanded Vbeta subsets, then cloned and sequenced skewed, oligoclonal, or monoclonal peaks.
Expanded Vbeta subsets were identified in almost all the patients. Over-represented Vbeta subsets from CD8-positive cells showed oligoclonal or monoclonal CDR3 size patterns. The CDR3 sequence repertoire in aplastic anaemia showed much redundancy compared with healthy donors. We identified patient-specific putative pathogenetic clonotypes that were not detectable in controls. In selected patients who were assessed longitudinally, these clonotypes were quantitatively related to disease activity. Selective killing of autologous haemopoietic progenitors by the Vbeta-specific lymphocyte population was shown in one patient. These apparently pathogenetic CDR3 sequences showed homology between individuals, suggesting a role for a "semi-public" immune response in the pathophysiology of aplastic anaemia.
In-vivo dominant clonal immune response can be identified in many patients with aplastic anaemia, which is evidence for an underlying antigen-driven immune process. Longitudinal tracking by molecular techniques could inform individual clinical decisions and the development of new treatments in autoimmune diseases.
Although the target of the aberrant immune response is the haemopoietic stem cell, the triggering antigens remain unknown. We combined cell phenotypic, molecular biology, and functional analyses to study the effector arm of immunity in an attempt to establish an immune pathophysiology. Clinical application of such a model could broadly extend to other autoimmune diseases.
The Lancet 01/2004; 364(9431):355-64. · 39.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: Aplastic anaemia in adults is usually acquired, but rarely constitutional types of bone marrow failure can occur late in life. We assessed two families with onset of pancytopenia in adults and detected two novel point mutations in the telomerase RNA gene (TERC) in each family. This gene is abnormal in some kindreds with dyskeratosis congenita. Individuals in our families with mutated TERC did not have physical signs of dyskeratosis congenita, and their blood counts were nearly normal, but all had severely shortened telomeres, reduced haemopoietic function, and raised serum erythropoietin and thrombopoietin. Bone marrow failure of variable severity due to dyskeratosis congenita, historically characterised by associated physical anomalies and early pancytopenia, may be present in otherwise phenotypically normal adults, and can masquerade as acquired aplastic anaemia.
The Lancet 12/2003; 362(9396):1628-30. · 39.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: The evolution of bone marrow failure syndromes such as aplastic anemia (AA) to clonal hematologic diseases such as myelodysplastic syndrome is well recognized. Cytogenetic abnormalities are commonly seen late events, particularly aneuploidy of chromosomes 7 and 8. A proportion of bone marrow failure patients may also develop aneuploidy that is detectable by fluorescence in situ hybridization but not by standard cytogenetic analysis. The molecular basis for aneuploidy in this setting is currently unknown but may include abnormalities in the mitotic spindle checkpoint. For this reason, we searched for mutations in the mitotic spindle checkpoint genes hBUB1 and hMAD2, and also examined the expression of hBUB1 in cells of bone marrow failure patients. No pathogenic mutations were found in 59 patients. Of 170 bone marrow failure patients, less than one-third expressed hBUB1 transcript. Gene expression profiling confirmed a significant down-regulation of hBUB1 message in patients. We conclude that mutations in mitotic spindle checkpoint genes do not account for aneuploidy in marrow failure states. However, we cannot exclude epigenetic inactivation of hBUB1 as a potential mechanism in some patients.
[show abstract][hide abstract] ABSTRACT: T cell-mediated suppression of haematopoiesis is believed to play an important role in the pathophysiology of aplastic anaemia (AA) and in the pancytopenia of some myelodysplastic syndromes (MDS). Natural-killer T (NKT) cells belong to a unique lymphocyte subset that expresses an invariant T-cell receptor (TCR), consisting of Valpha24JalphaQ, and common NK cell surface markers. NKT cells have been hypothesized to play a role in immune regulation, and many human autoimmune conditions are associated with NKT cell deficiency. Here we investigate the role of NKT cells in AA and MDS patients. Flow cytometry demonstrated that NKT cells, unlike other T-lymphocyte subpopulations, were disproportionally decreased in AA and MDS marrow. When we compared variability within the CDR3 region of Valpha24 in CD4-CD8- T cells derived from AA and healthy individuals, the CDR3 size of Valpha24 cells showed a polyclonal distribution in AA patients, while in control subjects a typical oligoclonal or monoclonal pattern was found. Southern blot and sequence analysis of Valpha24 polymerase chain reaction products revealed that the NKT cell-specific JalphaQ region was predominant in control subjects, whereas it was not, or only very weakly, detected in AA and MDS patients. These results show that NKT cells are profoundly decreased in AA and MDS, and their deficiency may, as in other human autoimmune diseases, play a role in the local immune dysregulation in AA and MDS.
British Journal of Haematology 01/2003; 119(3):803-9. · 4.94 Impact Factor
[show abstract][hide abstract] ABSTRACT: Bone marrow failure has been regarded as one of the triad of clinical manifestations of paroxysmal noctumal hemoglobinuria (PNH), and PNH in turn has been described as a late clonal disease evolving in patients recovering from aplastic anemia. Better understanding of the pathophysiology of both diseases and improved tests for cell surface glycosylphosphatidylinositol (GPI)-linked proteins has radically altered this view. Flow cytometry of granulocytes shows evidence of an expanded PNH clone in a large proportion of marrow failure patients at the time of presentation: in our large NIH series, about 1/3 of over 200 aplastic anemia cases and almost 20% of more than 100 myelodysplasia cases. Clonal PNH expansion (rather than bone marrow failure) is strongly linked to the histocompatability antigen HLA.-DR2 in all clinical varieties of the disease, suggesting an immune component to its pathophysiology. An extrinsic mechanism of clonal expansion is also more consistent with knock-out mouse models and culture experiments with primary cells and cell lines, which have failed to demonstrate an intrinsic proliferative advantage for PNH cells. DNA chip analysis of multiple paired normal and PIG-A mutant cell lines and lymphoblastoid cells do not show any consistent differences in levels of gene expression. In aplastic anemia/PNH there is surprisingly limited utilization of the V-beta chain of the T cell receptor, and patients' dominant T cell clones, which are functionally inhibitory of autologous hematopoiesis, use identical CDR3 regions for antigen binding. Phenotypically normal cells from PNH patients proliferate more poorly in culture than do the same patient's PNH cells, and the normal cells are damaged as a result of apoptosis and overexpress Fas. Differences in protein degradation might play a dual role in pathophysiology, as GPI-linked proteins lacking an anchor would be predicted to be processed by the proteasome machinery and displayed in a class I H.A. context, in contrast to the normal pathway of cell surface membrane recycling, lysosomal degradation, and presentation by class II HLA. The strong relationship between a chronic, organ-specific immune destructive process and the expansion of a single mutant stem cell clone remains frustratingly enigmatic but likely to be the result of interesting biologic processes, with mechanisms that potentially can be extended to the role of inflammation in producing premalignant syndromes.
International Journal of Hematology 09/2002; 76 Suppl 2:168-72. · 1.68 Impact Factor
[show abstract][hide abstract] ABSTRACT: Aplastic anemia (AA) remains an elusive disease. Its pathophysiology is not only fascinating by the seemingly simple findings of cytopenia and marrow hypoplasia, but may also contain key information to the understanding of other fundamental processes such as stem cell regeneration, evolution, and immune control of clonal diseases. Although measurements of blood counts provide an objective tool to assess the disease activity and response to the therapy, immune pathophysiology of AA, as inferred from the successes of immunosuppression, provides only few other clinical clues. Similarly, the current laboratory evidence remains mostly indirect. In spite of the recognition of immune pathways of hematopoietic inhibition and apoptosis in AA, the fundamental question about the nature of the antigen(s) inciting or maintaining the pathologic immune response that ultimately leads to bone marrow failure, remains open. However, recognition of the immune targets may aid in understanding not only the pathogenesis but also many of clinical associations and the late squelae of AA. For example, abnormal cells in AA and myelodysplastic syndrome (MDS) MDS may harbor inciting antigens but the immune response lacks selectivity. Clonal selection pressure may be a result of this process or alternatively, emergence of tolerance could lead to the establishment of abnormal hematopoiesis. Clonal proliferation of large granular lymphocytosis could represent an example of an exaggerated response to an immunodominant hematopoietic antigen. In addition to the traditional functional or phenotypic analysis, pathologic immune response in AA can be studied on molecular level by identifying and quantitating T cell clones based on the presence of unique variable B-chain CDR3 sequences. Detection of clonal expansion is based on the observation that in infections and autoimmune conditions, the presence of antigenic drive will lead to the expansion and overrepresentation of T cell clones recognizing this antigen. However, simple analysis of clonal representation is not sufficient to resolve the complex nature of the immune repertoire in the context of genetic and clinical heterogeneity. Therefore, we analyzed VB and CDR3 repertoire in CD4 and CD8 cells, activated or effector cell subsets. To distinguish truly expanded and likely immunodominant clones, we first studied VB distribution and cloned CDR3 sequences from expanded VB families. Identified clonotypic sequences can be used to design molecular tests to quantitate the strength of pathologic immune response. Clonotype sharing has been confirmed in patients with similar clinical features indicating presence of common antigens. In addition, quantitative analysis showed correlation with the therapy response. Persistence and patterns of clonotypes may be helpful in the classification of immune-mediated marrow failure based on the immune characteristics and will allow inferences into the inciting pathways.
International Journal of Hematology 09/2002; 76 Suppl 1:207-14. · 1.68 Impact Factor
[show abstract][hide abstract] ABSTRACT: We have hypothesized that in aplastic anemia (AA) the presence of antigen-specific T cells is reflected by their contribution to the expansion of a particular variable beta chain (V beta) subfamily and also by clonal CDR3 skewing. To determine the role of disease-specific "signature" T-cell clones in AA, we studied preferential V beta usage by flow cytometry and analyzed V beta-CDR3 regions for the presence of oligoclonality. We first established the contribution of each V beta family to the total CD4(+) and CD8(+) lymphocyte pool; in AA and paroxysmal nocturnal hemoglobinuria, a seemingly random overrepresentation of different V beta families was observed. On average, we found expansion in 3 (of 22 examined) V beta families per patient. When the contribution of individual V beta families to the effector pool was examined, more striking V beta skewing was found. V beta-CDR3 size distribution was analyzed for the expanded V beta families in isolated CD4(+) and CD8(+) populations; underrepresented V beta families displayed more pronounced CDR3 skewing. Expanded CD4(+)V beta subfamilies showed mostly a polyclonal CDR3 size distribution with only 38% of skewing in expanded V beta families. In contrast, within overrepresented CD8(+)V beta types, marked CDR3 skewing (82%) was seen, consistent with nonrandom expansion of specific CD8(+) T-cell clones. No preferential expansion of particular V beta families was observed, in relation to HLA-type. In patients examined after immunosuppressive therapy, an abnormal V beta-distribution pattern was retained, but the degree of expansion of individual V beta was lower. As V beta skewing may correlate with relative V beta size, oligoclonality in combination with numerical V beta expansion can be applied to recognition of disease-specific T-cell receptors.
[show abstract][hide abstract] ABSTRACT: Recently, phenotypically normal CD34 cells from the marrow of patients with paroxysmal nocturnal hemoglobinuria (PNH) were reported to show impaired growth and elevated Fas receptor expression as compared to glycophosphatidylinositol-anchored protein (GPI-AP)-deficient CD34 cells and CD34 cells from normal individuals. These results are consistent with the theory that PNH cells have an intrinsic growth advantage, but their superior expansion in vitro could also be the outcome of selective extrinsic pressure in vivo.
Growth characteristics, competitive features, and susceptibility to apoptosis of sorted normal or GPI-AP-deficient CD34(+) cells derived from PNH patients were assessed in suspension and methylcellulose cultures.
When we directly compared the growth of patients' CD34 cells, separated based on expression of GPI-AP CD55 and CD59, in most of the patients studied, mutant CD34 cells showed higher progeny production and outgrew phenotypically normal CD34 cells derived from PNH patients in mixing experiments. However, their proliferation rate did not exceed that of control CD34 cells. To determine whether deficient growth of phenotypically normal CD34 cells in PNH was secondary to a pre-existing in vivo insult, we determined the fraction of apoptotic cells within fresh normal and PNH CD34 cells. Normal CD34 cells from PNH patients showed a high proportion of apoptotic cells and higher Fas expression, while GPI-AP-deficient and control CD34 cells showed similar, low rates of apoptosis. After correction for pre-existing apoptosis, the proliferation potential of normal and PNH CD34 cells was similar.
These results strongly suggest that clonal expansion of GPI-AP-deficient progenitor cells from PNH patients is due to their selection in the hostile marrow environment of the patient.
[show abstract][hide abstract] ABSTRACT: We studied the degree and the pattern of skewing of the variable region of beta-chain (VB) T-cell receptor (TCR) repertoire in aplastic anemia (AA) at initial presentation and after immunosuppression using a high-resolution analysis of the TCR VB complementarity-determining region 3 (CDR3). Age-matched healthy individuals and multitransfused patients with non-immune-mediated hematologic diseases were used as controls. In newly diagnosed AA, the average frequency of CDR3 size distribution deviation indicative of oligoclonal T-cell proliferation was increased (44% +/- 33% vs 9% +/- 9%; P =.0001); AA patients with human leukocyte antigen (HLA)-DR2 and those with expanded paroxysmal nocturnal hemoglobinuria clones showed more skewed VB repertoires. Nonrandom oligoclonal patterns were found for VB6, VB14-16, VB21, VB23, and VB24 subfamilies in more than 50%, and for VB15, VB21, and VB24 in more than 70% of AA patients with HLA-DR2. Patients received immunosuppression with antithymocyte globulin (ATG)/cyclosporine (CsA) or cyclophosphamide (CTX) with CsA in combination, and their VB repertoire was reanalyzed after treatment. Whereas no significant change in the degree of VB skewing in patients who had received ATG was seen, patients treated with CTX showed a much higher extent of oligoclonality within all VB families, consistent with a profound and long-lasting contraction of the T-cell repertoire. VB analysis did not correlate with the lymphocyte count prior to lymphocytotoxic therapy; however, after therapy the degree of VB skewing was highly reflective of the decrease in lymphocyte numbers, suggesting iatrogenic gaps in the VB repertoire rather than the emergence of clonal dominance. Our data indicate that multiple specific clones mediate the immune process in AA.
[show abstract][hide abstract] ABSTRACT: We hypothesized that an active autoimmune process in aplastic anemia (AA) corresponds to the expansion of cytotoxic lymphocytes (CTLs) displaying mature effector phenotype. We determined whether the numbers of effector CTLs in blood of patients with bone marrow failure syndromes are elevated and correlate with the disease activity and responsiveness to immunosuppression.
We analyzed samples from patients with AA, myelodysplastic syndrome (MDS), polytransfused patients with nonimmune-mediated hematologic disease, and normal controls for the presence of effector T lymphocytes using four-color flow cytometry. Expression of CD57 and loss of CD28 on CD8+CD3+ CTL were used as markers for the terminal effector phenotype. In addition, intracellular staining for perforin and granzyme B was preformed. The numbers of effector CTL did not differ between healthy individuals and hematologic controls and the two groups were pooled.
The percentages of CD8+CD28- and CD8+CD28-CD57+ cells were significantly higher in AA and MDS patients than in controls. There was a trend toward a gradual decrease in the effector CTLs from the high values observed in untreated new patients and patients who did not respond to immunosuppression, intermediate levels for partial responders and complete responders, to the lowest levels seen in controls. However, severity of pancytopenia did not correlate with the size of the effector cell population. In contrast to CD57+ CTLs, expression of perforin or granzyme B in the cytotoxic effector cells did not differ in AA patients from those of controls.
Our results indicate that phenotypically defined effector CTLs are increased in AA and MDS and the effector phenotype may be useful to isolate and characterize antigen-specific T cells in AA in order to delineate the possible inciting or driving agents in AA.
[show abstract][hide abstract] ABSTRACT: Immune mediation of aplastic anemia (AA) has been inferred from clinical responsiveness to immunosuppressive therapies and a large body of circumstantial laboratory evidence. However, neither the immune response nor the nature of the antigens recognized has been well characterized. We established a large number of CD4 and CD8 T cell clones from a patient with AA and analyzed their T cell receptor (TCR) usage. Most CD4 clones displayed BV5, whereas most CD8 clones displayed BV13. We found sequence identity for complementarity determining region 3 (CDR3) among a majority of CD4 clones; the same sequence was present in marrow lymphocytes from four other patients with AA but was not detected in controls. The dominant CD4 clone showed a Th1 secretion pattern, lysed autologous CD34 cells, and inhibited their hematopoietic colony formation. In three of four patients, successful immunosuppressive treatment led to marked decrease in clones bearing the dominant CDR3 BV5 sequence. These results suggest surprisingly limited heterogeneity of the T cell repertoire in an individual patient and similarity at the molecular level of the likely pathological lymphocyte response among multiple patients with AA, consistent with recognition of limited numbers of antigens shared by individuals with the same HLA type in this disease.
Journal of Clinical Investigation 10/2001; 108(5):765-73. · 12.81 Impact Factor