Destruction Of Hematopoietic Stem Cells Research Articles

Research progress of cyclosporine therapy for peri-pregnancy aplastic anemia patients

The pathogenesis of aplastic anemia(AA) is extremely complicated. The immune-mediated destruction of hematopoietic stem cells has been considered the main pathophysiological mechanism of AA. Successful pregnancy of AA patients are possible. More than 50% of them have good pregnancy outcomes. Nevertheless, maternal and infant′s complications in AA patients with pregnancy are very common. It is safe to use cyclosporine A to treat AA in pregnancy. However, the risk of maternal and infant′s complications would be increased. It is essential that the AA patients who is taking cyclosporine A should be monitored closely and frequently throughout pregnancy. Once severe drug side effects appear without control, discontinue cyclosporine A or terminate pregnancy. Cyclosporine A can be excreted into breast milk, while the ingestion amount of cyclosporine A through breast feeding and the levels of serum cyclosporine A are relatively low in most infants. While there is a lack of information about long-term outcomes, the safety of taking cyclosporine A while breast-fed is still controversial. As a result, when advising patients regarding breast-feeding, the advantages should be balanced with the minimal risk of exposure to cyclosporine A in breast milk. Key words: Anemia, aplastic; Cyclesporine; Peripartum period

Clonal evolution and clinical significance of copy number neutral loss of heterozygosity of chromosome arm 6p in acquired aplastic anemia

Acquired aplastic anemia (aAA) results from the T cell-mediated autoimmune destruction of hematopoietic stem cells. Factors predicting response to immune suppression therapy (IST) or development of myelodysplastic syndrome (MDS) are beginning to be elucidated. Our recent data suggest most patients with aAA treated with IST develop clonal somatic genetic alterations in hematopoietic cells. One frequent acquired abnormality is copy-number neutral loss of heterozygosity on chromosome 6p (6p CN-LOH) involving the human leukocyte antigen (HLA) locus. We hypothesized that because 6p CN-LOH clones may arise from selective pressure to escape immune surveillance through deletion of HLA alleles, the development of 6p CN-LOH may affect response to IST. We used single nucleotide polymorphism array genotyping and targeted next-generation sequencing of HLA alleles to assess frequency of 6p CN-LOH, identity of HLA alleles lost through 6p CN-LOH, and impact of 6p CN-LOH on response to IST. 6p CN-LOH clones were present in 11.3% of patients, remained stable over time, and were not associated with development of MDS-defining cytogenetic abnormalities. Notably, no patient with 6p CN-LOH treated with IST achieved a complete response. In summary, clonal 6p CN-LOH in aAA defines a unique subgroup of patients that may provide insights into hematopoietic clonal evolution.

Establishing aplastic anemia animal model based on different pathogenesis

Aplastic anemia(AA) is a disease,including congenital AA and acquired AA, characterized by an extremely hypocellular marrow and peripheral blood pancytopenia due to bone marrow failure. Congenital AA is a autosomal recessive disease due to gene mutation. Persently, acquired AA is recognized as a disease caused by destruction of hematopoietic stem cells, defective marrow microenvironment and aberrant T cellular-immunity. In order to further study its pathogenesis and to choose effective therapeutic target, it has important clinical significance to establish correspondent animal model based on different pathogenesis. This article summarizes the congenital AA amimal models including Fanc A(-/-) mouse, Fanc C(-/-) mouse, Fanc G(-/-) mouse, Fanc D1(-/-)/Fanc 2(-/-) mouse, Fanc D2(-/-) mouse and other gene deficiency mouse AA models, and the acguired AA models resulting from the hematopoietic stem cell decrease, hematopoietic microenvironment injury, immune mediation and combination of hematopoietic stem cell dicrease with immune mediation.

Clonal Evolution and Clinical Significance of Copy Number Neutral Loss of Heterozygosity of Chromosome Arm 6p in Acquired Aplastic Anemia

Acquired Aplastic Anemia (aAA) develops from the immune-mediated destruction of hematopoietic stem cells (HSCs) by auto-reactive T cells. Although the exact mechanisms remain incompletely defined, it is hypothesized that auto-reactive T cells target auto-antigens presented by human leukocyte antigens (HLA), leading to HSC destruction. Acquired genetic alterations resulting in clonal hematopoiesis may therefore emerge in patients with aAA as a means of evading this immune response. Well-recognized patterns of clonal cellular evolution in aAA include myelodysplastic syndrome (MDS) and paroxysmal nocturnal hemoglobinuria (PNH). Recent studies have identified acquired copy-number neutral loss of heterozygosity on the short arm of chromosome 6 (6p CN-LOH) as another recurrent abnormality. The HLA loci map to 6p, and deletions or LOH in this region result in the loss of alleles from one parental HLA haplotype. 6p CN-LOH may allow for immune escape and restoration of hematopoiesis in aAA by deleting the HLA haplotype to which the HLA-restricted auto-reactive T cell response is targeted. The effect of 6p CN-LOH on clinical outcome and further clonal evolution in aAA is not well described. Thus, the aim of our study was to investigate the frequency, evolution and clinical significance of clonal 6p CN-LOH in our institutional cohort of adult and pediatric patients diagnosed with aAA.Bone marrow (BM) or peripheral blood (PB) samples were collected for genome wide single nucleotide polymorphism (SNP) arrays (Illumina Human OMNI1 or 850K BeadChip), and analyzed for the presence of copy number abnormalities and loss of heterozygosity, with specific reference to 6p CN-LOH. Clinical outcome data were extracted from patient charts. High Resolution HLA typing information was obtained from patient charts or performed on a research basis. HLA allele frequency in our cohort was compared to that of published control populations from the National Marrow Donor Program. Quantitative comparison of HLA haplotype allele frequency in patients with 6p CN-LOH was performed by next generation sequencing to determine the identity of clonally deleted parental alleles.Our cohort was comprised of 71 patients with aAA and related conditions including PNH and hypoplastic MDS (AA (n=64), PNH (n=5), MDS (n=2); median age 14 years, range 8 months to 68 years; 52% females). A total of 68 patients (96%) had a SNP array performed. 6p CN-LOH (n=8) or acquired clonal deletion of 6p (n=1) was detected in 13% of these patients, with the prevalence significantly higher in adult versus pediatric patients (29% versus 8.3%, p < 0.045). In all patients, clones were first identified at least 6 months after initial diagnosis. The majority of patients with 6p CN-LOH (n=7) received immunosuppressive therapy (IST). Of these, 43% failed IST requiring additional therapy while the rest achieved only a partial response. Importantly, no patient with 6p CN-LOH receiving IST alone achieved complete remission at the time of analysis. The characteristics of 6p CN-LOH varied considerably in terms of the number of clones with different sized regions of LOH, the percentage of cells with LOH, and the specific HLA loci included in the LOH region. 6p CN-LOH patients in whom follow-up SNP analyses were performed exhibited stable clone size, with no new clonal abnormalities. Quantitative analysis of HLA alleles in 6p CN-LOH patients revealed many distinct functionally deleted HLA alleles. While no specific allele was recurrently lost in the 6p CN-LOH clones, several of these deleted alleles occurred at increased frequency in our overall aAA cohort compared to ethnicity-matched control populations.In conclusion, the results from our institutional aAA cohort confirm the prevalence rates of clonal 6p CN-LOH described previously in ethnically distinct populations, while demonstrating an increased frequency of 6p CN-LOH in adult versus pediatric aAA patients. None of the aAA patients with 6p CN-LOH in our cohort achieved a complete response to IST alone, suggesting that 6p CN-LOH may be a risk factor or marker predicting poor response to IST. Taken together, our findings underscore the critical importance of understanding the mechanisms underlying clonal evolution in aAA and their effects on disease pathogenesis and response to therapy. Future multi-institutional studies are needed to define optimal therapeutic approaches for aAA patients with 6p CN-LOH. DisclosuresNo relevant conflicts of interest to declare.

Aplastic anemia as a feature of systemic lupus erythematosus: a case report and literature review

Peripheral cytopenias are common in systemic lupus erythematosus, but bone marrow involvement is rarely reported. Aplastic anemia is the result of immune-mediated destruction of hematopoietic stem cells causing pancytopenia and characterized by an empty bone marrow. This rare but serious disease has been described as an unusual manifestation of systemic lupus erythematosus. We reviewed the 25 cases published in the English language literature and discuss the clinical presentation, outcome, treatment, and pathophysiology of aplastic anemia as a complication of systemic lupus erythematosus. We report here the first case of aplastic anemia associated with systemic lupus erythematosus treated with an allogeneic hematopoietic stem cell transplant. Over one half of patients received concomitantly the diagnoses of systemic lupus erythematosus and aplastic anemia. No clinical or histological features can distinguish primary aplastic anemia from aplastic anemia occurring in systemic lupus erythematosus patients. The overall mortality is about 15% and corticosteroid-based therapy alone or in combination with other immunomodulatory drugs can restore bone marrow function. Systemic lupus erythematosus may be complicated by bone marrow involvement. The diagnosis of peripheral cytopenias should be confirmed by bone marrow aspiration. All these patients should receive cortisone as a first treatment. Plasma exchanges seem to have some efficacy. Other different immunomodulatory therapies were used with variable results.

New therapeutic approaches for protecting hematopoietic stem cells in aplastic anemia

Aplastic anemia (AA) is an immune-mediated and life-threatening form of acquired bone marrow failure (BMF), characterized by development and expansion of self-reactive T cells. These T cells cause continuous destruction of hematopoietic stem cells (HSCs), progenitors, and mature blood cells, leading to severe and if left untreated fatal marrow hypoplasia and pancytopenia. Standard treatment options for patients with AA include: (1) immunosuppressive therapy (IST) with anti-thymocyte globulin and cyclosporine A which targets self-reactive T cells, or (2) matched sibling or unrelated BM transplant (BMT). The IST treatment is often not effective due to poor response to therapy or disease relapse after IST. Also, BMT is not an option for many patients due to their age, comorbidities, and the lack of histocompatible donor. This necessitates development and testing of novel approaches to reduce severity of AA and to efficiently treat patients with refractory and relapsed AA. Immune-mediated AA was reproduced in animals, including mouse lymphocyte infusion models, which are used to study further etiology and pathophysiology of AA and test new drugs and approaches in treating and managing AA. In these mouse models the immune correlates and pathologic features of AA are strikingly similar to features of severe human AA. In this article we (a) briefly review standard and developing approaches for treating AA and (b) describe development and testing of novel treatment approach with a potential to safely reduce BM hypoplasia and significantly decrease the loss of HSCs in mouse lymphocyte infusion model of AA.

Immunologically-Selected Hematopoiesis Caused by HLA Allelic Loss In Patients with Aplastic Anemia

AbstractAbstract 198 Background:Idiopathic aplastic anemia (AA) is a syndrome characterized by pancytopenia and bone marrow (BM) hypoplasia, which are caused by the auto-immune destruction of hematopoietic stem cells (HSCs). However, little is known about the nature of HSCs that survive the occurrence of autoimmune insults and maintain hematopoiesis both during and after aplastic diathesis. While the severity of the autoimmunity is the major determinant of BM failure in patients with AA, some intrinsic genetic changes in HSCs could also be involved in the disease process, since the clonality of the residual, persistent hematopoiesis under the aplastic state has been well recognized. Objectives/Methods:To characterize the nature of the HSCs that support hematopoiesis in AA, peripheral blood (PB) specimens obtained from 317 patients with AA were subjected to the genome-wide analysis of genetic lesions using Affymetrix® 500K SNP arrays. For the normal controls, 1746 PB specimens from the Japan Marrow Donation Program (JMDP) were also analyzed. All specimens were genotyped for HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 alleles. In the eligible cases, PB leukocytes and CD34+ BM cells were also examined to determine their expression of HLA-A antigens using allele-specific monoclonal antibodies by flow cytometry (FCM). To identity the common HLA types associated with AA, a total of 6,629 registries from JMDP who had received allogeneic bone marrow transplantation between 1992 and 2008 were employed, where the HLA frequencies in AA (N=406) were compared with those among other hematopoietic disorders, in acute myeloid leukemia (N=1,822), acute lymphocytic leukemia (N=1,406), chronic myeloid leukemia (N=1,014), myelodysplastic syndromes (N=824), non-Hodgkin's lymphoma (N=565), and other neoplastic disorders (N=392). Results:A number of genetic alterations were detected in our AA case series, among which the most conspicuous was acquired uniparental disomy (UPD), or the copy number-neutral loss of heterozygosity, involving the 6p arms (6pUPD). The 6pUPD was identified in 38 patients (12%) but not in any JMDP donor specimens, and it commonly affected the HLA locus, which was expected to result in the loss of one HLA haplotype. In fact, loss of HLA-A expression from the missing haplotype was confirmed by FCM in all 13 6pUPD-positive cases thus far tested, whereas the HLA-A expression from both haplotypes was preserved in the 58 samples without 6pUPD. The loss of HLA-A expression in the 6pUPD-positive cases was found in multiple lineages of leukocytes, including granulocytes, monocytes, B cells, BM CD34+ cells, and to a lesser extent in T cells. Of particular interest was the fact that the missing HLA haplotypes that were predicted from the SNP array data were extremely biased to particular class I HLA alleles, including HLA-A*02:01, HLA-A*02:06, A*31:01, B*40:02, and B*40:06. Moreover, when the frequencies of these alleles were compared among the 6,629 JMDP registries, they were shown to be strongly associated with AA in comparison to other non-significant HLA alleles, where the odds ratios for these alleles with regard to non-significant alleles as for the risk of the development of AA were 2.00 (95%CI; 1.52 – 2.62) for A*02:01, 2.37 (95%CI; 1.80 – 3.12) for A*02:06, 1.46 (95%CI; 1.06 – 2.02) for A*31:01, 2.07 (1.56 - 2.77) for B*40:02, and 2.67 (1.95 - 3.66) for B*40:06. Conclusions:AA patients frequently show permissive hematopoiesis with 6pUPD, which is thought to develop due to the occurrence of auto-immune insult based on the Darwinian principle of “survival of the fittest” (Figure). The tight association of AA with particular class I antigens that are invariably missing in permissive hematopoiesis with 6pUPD strongly supports the hypothesis that the auto-immunity responsible for AA is primarily mediated by cytotoxic T cells which target a relatively limited species of auto-antigens presented on HSCs through these relevant HLAs. Our findings therefore also provide a solid basis for isolating the target auto-antigens responsible for the development of AA in the future studies. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Effects of Rabbit Anti-Mouse Thymocyte Globulin Treatment in a Splenocyte-Induced Model of Aplastic Anemia

AbstractAbstract 1172Acquired aplastic anemia is an immune-mediated disease where destruction of hematopoietic stem cells (HSCs) in the bone marrow results in severe and life-threatening pancytopenia. Thymoglobulin® is often used as immunosuppressive therapy in this disease with up to 80 percent of patients responding to a combination of Thymoglobulin and cyclosporine. In an effort to better understand the activities and mechanism of action of Thymoglobulin we developed a mouse model of immune-mediated aplastic anemia and evaluated a murine surrogate of Thymoglobulin®, rabbit anti-mouse thymocyte globulin (mATG) in this model. We modified a graft-versus-host (GVH)-induced model described in the literature (Bloom, et al., 2004) utilizing HSC-depleted spleen cells transferred from C57BL/6 into CByB6F1 mice instead of lymph node cell transfer. Our modified model shows a cell dose-dependent increase in pancytopenia and lethality. Mice receiving a high dose (100×106) of HSC-depleted splenocytes experienced severe pancytopenia and rapid death occurring around day 21 whereas mice receiving lower doses (70×106, 35×106 and 17×106) of cells showed progressively less pancytopenia and lethality as the dose of cells decreased. Histopathology also showed marked loss of hematopoietic progenitor cells in the bone marrow with little evidence of GVHD in other tissues. Prophylactic administration of mATG (25mg/kg, 2x, day 0 and 3) to mice given high doses of HSC-depleted splenocytes (100×106) resulted in a significant improvement in pancytopenia and survival (70%) in this model. Interestingly, therapeutic administration of mATG was more effective when given later relative to disease induction. Delivery of mATG (25mg/kg, 2x, three days apart) starting on day 3 showed some delay in disease progression (day 30 vs day 21) and mATG started on day 6 slightly increased survival (40%). However, mice receiving mATG starting on days 10 or 14 showed a much greater overall survival of 100% and 60%, respectively, with full rebound of hematopoietic cells in the blood to normal levels. The complete response observed with later mATG administration (day 10 or day 14) mimics the treatment and response of patients given Thymoglobulin®. In summary, we have established a novel model of HSC-depleted splenocyte induction of bone marrow failure in mice that is responsive to therapeutic ATG administration. Studies in this model will aid in further understanding the mechanism of ATG in aplastic anemia and may contribute to the development of potential new therapies. Disclosures:Ruzek:Genzyme: Employment. Phillips:Genzyme: Employment. Richards:Genzyme: Employment. Mamlouk:Genzyme: Employment. Williams:Genzyme: Employment. Garman:Genzyme: Employment.

Aplastic anemia.

Most acquired aplastic anemia is the result of immune-mediated destruction of hematopoietic stem cells causing pancytopenia and an empty bone marrow, which can be successfully treated with either immunosuppressive therapy or hematopoietic stem-cell transplantation. In aplastic anemia, oligoclonally expanded cytotoxic T cells induce apoptosis of hematopoietic progenitors. T-bet, a transcription factor that binds to the interferon-gamma promoter region, is upregulated in aplastic anemia T cells. Regulatory T cells are significantly reduced in patients' peripheral blood and in an aplastic anemia murine model, infusion of regulatory T cells ameliorates disease progression. In a minority of cases, loss-of-function mutations in telomerase complex genes may underlie disease development. Long-term survival, once strongly linked to response to immunosuppressive therapy, can now be achieved even among nonresponders due to significant advances in supportive care and better salvage treatments. Evidence has accumulated in the recent years further corroborating an immune-mediated process underlying aplastic anemia pathogenesis. Hematopoietic stem-cell transplantation from a matched sibling donor is preferred for children and young adults with severe aplastic anemia, and immunosuppressive therapy is employed when hematopoietic stem-cell transplantation is not feasible due to age, lack of a histocompatible sibling, co-morbidities, or by patient choice.

Decreased Numbers of Tregs in Aplastic Anemia Is Detected by Immunohistochemistry and Flow Cytometry.

Idiopathic aplastic anemia (AA) is characterized by immune-mediated destruction of hematopoietic stem cells, leading to peripheral pancytopenia. Immune pathogenesis in AA is supported by experimental data, as well as clinical observations and may be related to the breach of peripheral or central tolerance. Regulatory T cells (Treg) constitute one of the most important mechanisms of central tolerance engaged in the down-modulation of autoreactive T cells. Tregs have been found to be reduced in several autoimmune diseases and decreased frequencies of Tregs were also reported in AA and MDS. Overexpression of the high affinity IL-2 receptor alpha chain (CD25) and the forkhead family transcription factor P3 (FoxP3), required for the development and function of Tregs, serve as phenotypic markers for Tregs. We investigated Treg levels in a cohort of AA patients (N=21) and healthy individuals (N=15); flow cytometric quantification of Treg was carried out after surface/intracellular staining of whole blood for Treg markers (CD3, CD4, CD25, FoxP3). After proper gating (light scatter properties, CD3, CD4, CD25), CD4+ T cells were subdivided into CD25−, CD25int and CD25hi populations, and the co-expression of CD25hi and Foxp3 was analyzed. In comparison to controls, AA patients (N=12) show not only lower frequencies of CD4+CD25hi+ T cells within the total lymphocyte population (median 0.07% vs. 0.21%; p=.03), but also absolute lower absolute numbers (1.31/uL vs. 5.78/uL, p=.0002). Similarly, CD4+CD25hi+FoxP3+ T cells were found to be depressed in untreated AA patients in comparison to controls (median 0.07% vs. 0.21% and 1.06/uL vs. 4.76/uL; p=.03 and p=.003). While Tregs were lower in patients with active disease unresponsive to immunosuppressive treatment (responder 0.1% vs non responder 0.07%, CD4+CD25hi Tcells, p=.02), serial testing performed in 6 patients treated with ATG/CsA did not reveal correlation between hematologic improvement and recovery of Treg numbers. When double immunohistochemical staining for CD3 and Foxp3 was performed in pre-treatment bone marrow core biopsies of AA patients (N=3) and controls (N=2) a mean of 3 CD3+Foxp3+ cells/10 high power fields (hpf) were counted (vs. mean 28/10 hpf, p<.05 in controls), suggesting that lower numbers of Tregs were also present in the bone marrow of AA patients. In conclusion, our results suggest that Tregs are decreased in blood and marrow of patients with idiopathic AA, consistent with the breach of peripheral tolerance in AA. In addition to flow cytometry, immunohistochemical staining of histologic specimens can be used for the quantitative analysis of Tregs in bone marrow failure syndromes and other immune-mediated conditions such as GvHD.

Perforin gene mutations in patients with acquired aplastic anemia

AbstractPerforin is a cytolytic protein expressed mainly in activated cytotoxic lymphocytes and natural killer cells. Inherited perforin mutations account for 20% to 40% of familial hemophagocytic lymphohistiocytosis, a fatal disease of early childhood characterized by the absence of functional perforin. Aplastic anemia, the paradigm of immune-mediated bone marrow failure syndromes, is characterized by hematopoietic stem cell destruction by activated T cells and Th1 cytokines. We examined whether mutations in the perforin gene occurred in acquired aplastic anemia. Three nonsynonymous PRF1 mutations among 5 unrelated patients were observed. Four of 5 patients with the mutations showed some hemophagocytosis in the bone marrow at diagnosis. Perforin protein levels in these patients were very low or absent, and perforin granules were completely absent. Natural killer (NK) cell cytotoxicity from these patients was significantly decreased. Our data suggest that PRF1 genetic alterations help explain the aberrant proliferation and activation of cytotoxic T cells and may represent genetic risk factors for bone marrow failure.

Cytokine gene polymorphisms in acquired bone marrow failure

Some acquired aplastic anemia (AA) results from immune-mediated destruction of hematopoietic stem cells. Cytokine gene polymorphisms are implicated in controlling cytokine production and increasing the susceptibility to some autoimmune diseases. We characterized the IL-6/-174, TNF-alpha/-308, IL-10/-1082, IFN-gamma/+874, TGFbeta1/-509 single nucleotide polymorphisms (SNP's) and the IL1-RA second intron variable number tandem repeat (VNTR) alleles in 73 patients with AA and compared the frequency of genotypes to established control populations. We found that some patients with acquired AA have polymorphisms which are linked to high production of proinflammatory cytokines, particularly TNF-alpha and IFN-gamma.

Perforin Gene Mutations in Patients with Acquired Aplastic Anemia.

Perforin is a cytolytic protein expressed mainly in activated cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. In T and NK cells perforin is stored in cytoplasmic granules and is essential for killing via non-Fas-mediated mechanisms. Perforin regulates the translocation of granzyme B from cytotoxic cells into target cells; after entering the target-cell granzyme B migrates to the target cell nucleus to participate in triggering apoptosis. Functional perforin is essential for normal CTL and NK cell function; without perforin CTL and NK cells show reduced or no cytolytic effect. Inherited perforin mutations account for 20–40% of familial hemophagocytic lymphohistiocytosis, an autosomal recessive fatal disease of early childhood characterized by uncontrolled accumulation of activated T cells and macrophages in many organs, increased Th1 cytokines and absent functional perforin. Acquired aplastic anemia (AA), the paradigm of immune mediated bone marrow failure syndromes, is characterized by hematopoietic stem cell destruction by activated T cells and Th1 cytokines. We examined whether mutations in Prf1 occur in AA; peripheral blood DNA samples from 75 patients and 302 controls were analyzed. Three novel nonsynonymous Prf1 mutations among five unrelated patients (ages: 21, 31, 33, 75, and 77 years old), not present in controls, were discovered; two polymorphisms were also identified (H300H, A274A). The mutations were in the coding region of Prf1 gene. In exon 2, arginine was replaced by histidine in one patient (CGT/CAT, R4H) and in 3 patients the same A91V mutation was identified (GCG/GTG, alanine to valine substitution). In exon 3, serine was replaced by isoleucine (S388I; AGC/ATC) in one patient. Germ-line origin of the Prf1 mutations was established by their presence also in DNA from buccal mucosa obtained from affected AA patients. Four of five patients with mutations showed some hemophagocytosis in the bone marrow examination when first diagnosed, but there were no other typical features of hemophagocytic syndrome such as hepatosplenomegaly or altered liver function tests. None of the patients with Prf1 mutations experienced hematologic recovery with immunosuppressive treatment. Perforin protein levels in all patients carrying mutations were very low or absent. By confocal microscopy, CD8 cells from patients with Prf1 mutations had complete absence of perforin granules (perforin and cathepsin D showed the expected pattern of co-localization in controls' cytotoxic granules). NK cell killing efficiency from patients carrying mutations in a standard Cr51-release cytolytic assay was significantly decreased compared to controls. Prf1 gene mutations may be related to a more severe phenotype of AA associated with marrow hemophagocytosis and failure to respond to immunosuppression. Mutations in the immune regulatory mechanisms identified in young children can manifest in adults without typically associated clinical findings or a suggestive family history. Mechanistically, Prf1 gene mutations help explain the aberrant proliferation and activation of cytotoxic T cells that are destructive of hematopoietic stem cells in AA and may be useful as predictive factors for responses to immunosuppressive treatments and the decision to rapidly undertake stem cell replacement. Prf1 gene mutations are genetic risk factors for bone marrow failure syndromes.

FOXP3-Positive Regulatory T-Cells in Acquired Aplastic Anemia.

CD4+CD25+ regulatory T cells (Treg) have been hypothesized to control the development and progression of autoimmunity by suppressing autoreactive T cells. Treg are characterized by constitutive expression of membrane CD25 and intracellular expression of the transcription factor forkhead box (FOX) P3. FOXP3 and NF-AT1 have key roles in regulatory T-cell development and function: induction of FOXP3 in naïve T cells by gene transfer results in gain of a regulatory phenotype, and NF-AT1 modulates transcription of FOXP3. Treg display their suppressive properties on CD4+ CD25- T cells when activated via the T cell receptor or Toll-like receptor-2 (TLR2). Decreased numbers of FOXP3-positive regulatory Tregs have been associated with impaired immune homeostasis. Treg numbers are deficient in patients with active systemic lupus erythematosus, GVHD, and autoimmune hepatitis; in multiple sclerosis, decreased FOXP3 impairs Treg function. Acquired aplastic anemia (AA), the paradigm of immune mediated bone marrow failure syndromes, is characterized by immune-mediated destruction of hematopoietic stem cells. To examine expression of CD4+CD25+ T-cells in this disease, peripheral blood mononuclear cells from AA patients, sampled at diagnosis and prior to immunosuppressive therapy (n=18), were examined by flow cytometry: CD4+CD25hi+ T-cells were markedly reduced or absent in patients compared to healthy controls (n = 12, 0.08± 0.01 vs 0.4± 0.1%, p=0.001). CD4+CD25hi+FOXP3+ T-cells were also significantly lower in patients (0.05± 0.01 vs 0.32± 0.1, p=0.001). By immunoblot, we observed significantly decreased and often undetectable FOXP3 protein levels in CD4+CD25+ T cells from patients (n=8) compared to controls (n=6). Low FOXP3 protein levels correlated with decreased FOXP3 mRNA levels, as measured by RT-PCR and quantitative real-time PCR experiments (n=5, p=0.03). There were no differences in TLR2 in immunoblots between patients and healthy controls despite differences in FOXP3 expression. Patients (n=8) with low FOXP3 levels also showed decreased or absent NF-AT1 protein levels. These data collectively implicate a transcriptional mechanism for FOXP3 down-regulation. In confocal microscopy, purified CD4+CD25+ T cells from patients (n=5) showed undetectable NF-AT1 and FOXP3 levels as compared to controls (n=3). Four patients studied 3–6 months after first sampling and post-immunosuppressive treatment showed increased Tregs and FOXP3 expression; four further patients in complete remission after immunosuppressive treatment had increased CD4+CD25hi+ T-cells as compared to treatment-naïve patients. CD4+CD25+FOXP3+ regulatory T-cells are decreased in most patients with AA, probably through transcriptional regulation; decreased NFAT1 could explain low FOXP3 expression and Treg frequency. Treg employed as cellular therapy in a murine model of immune-mediated AA can prevent T cell-mediated marrow destruction (Chen J and Young NS, unpublished data). Treg defects are now implicated in autoimmune marrow failure in aplastic anemia.

T-bet Over-Expression in T Cells from Patients with Aplastic Anemia.

Acquired aplastic anemia (AA) is a bone marrow failure syndrome characterized by immune-mediated destruction of hematopoietic stem cells. T cells from patients with AA overproduce IFN-γ, a cytokine that inhibits hematopoietic stem cell proliferation and induces Fas-mediated apoptosis; stem cell depletion results in marrow hypoplasia and peripheral blood pancytopenia. In T cells, regulation of IFN-γ production occurs primarily at the level of transcription. A proximal site of the IFN-γ gene (−75 to −45bp of the IFN-γ promoter) is a binding site for different transcription factors including NFAT, AP-1, ATF, CREB, and T-bet. T-bet is a member of the T-box family of transcription factors, this family contains a highly conserved DNA binding domain, the T-box, that binds to a specific sequence in the promoter of different genes, including the IFN-γ promoter. T-bet is found in Th1 but not in Th2 cells and is the key regulator of Th1 development and function (Rengarajan et al., Immunol Today 2000; 21: 479). The inducible expression of T-bet is mediated in part by Itk kinase (Sjabo et al., Science 2005; 307: 430). In the present study, we examined T-bet protein levels in T cells from patients with AA. Samples from 17 of 20 patients examined (85%) by immunoblot showed increased T-bet protein levels in unstimulated T cells compared to normal controls (p = 0.0001). Normal controls showed undetectable T-bet protein levels in unstimulated T cells but T-bet expression was induced after 24 hrs of stimulation with PMA and ionomycin. In electrophoretical mobility shift assays, we observed increased T-bet binding to the proximal site of the IFN-γ promoter in T cells from patients with AA; no binding was detected in unstimulated T cells from healthy controls, but binding was present after stimulation for at least 24 hrs. T-bet protein levels correlated with disease activity. Patients with increased T-bet protein levels showed increased intracellular IFN-g levels compared to controls, as detected by flow cytometry (p<0.05). Patients that expressed increased T-bet protein levels also showed increased levels of the Itk kinase (p=0.02). We examined if other kinases that lie downstream of Itk in the signal transduction activation cascade in T cells affected the inducible expression of T-bet. In normal T cells, rottlerin, a PKC-theta (PKC-θ) inhibitor, decreased T-bet protein levels by 50%; in AA T cells, rottlerin also decreased T-bet protein levels and IFN-γ intracellular levels by 50%. Our results suggest that the increased IFN-γ levels observed in AA are the result of activation of transcription of the IFN-γ gene by the regulator T-bet. Blocking of transcription of the IFN-γ gene by kinase inhibitors might represent a therapuetic strategy for AA and other human T cell-mediated autoimmune diseases.

Decreased TCR ζ-Chain Expression in T Cells from Patients with Aplastic Anemia.

The ζ-chain is a unique component of the T cell receptor (TCR) with no sequence homology to other CD3 molecules. The ζ-chain acts to stabilize the TCR on the cell surface and to transmit signals to different transduction pathways in the cell. T cells from patients with autoimmune diseases, with chronic inflammation, and suffering malignant tumors are functionally impaired in that they express decreased ζ-chain. Acquired aplastic anemia (AA) is a bone marrow failure syndrome characterized by T-cell immune mediated destruction of hematopoietic stem cells. Oligoclonal expansion of T cells correlates with disease activity. In this study, we examined TCR ζ-chain protein levels in aplastic anemia patients. Twelve out of sixteen patients (75%) examined displayed decreased ζ-chain protein levels compared to normal controls (p=0.008). As a control, no differences were detected in expression of the TCR ε-chain. Patients and healthy donors displayed comparable amounts of Zap-70, the kinase responsible for full phosphorylation and activation of the ζ-chain. There was no correlation with disease status or medical treatment at the time of sampling. Four patients were re-examined for ζ-chain protein levels at 3 month intervals, but there was no change in their ζ-chain expression pattern despite hematologic improvement. We hypothesized correlation between ζ-chain and IFN-g levels in T cells from patients with AA. When we estimated IFN-g levels by flow cytometry, all 5 patients examined had increased IFN-g levels, of whom 4 had decreased ζ-chain levels: intracellular IFN-g was reciprocally correlated with ζ-chain protein levels. In summary, our data indicate that TCR ζ-chain expression is decreased in the majority of patients with AA, regardless of disease activity or treatment status. ζ-chain-deficient T cells from AA patients also overproduce IFN-g. Is ζ-chain down-regulation the primary event leading to IFN-g over-production, or is IFN-g responsible for the ζ-chain down-regulation? One possible explanation is an intrinsic defect in the TCR leading to alterations in kinases and transcription factors downstream of the ζ-chain-Zap-70 association. Whether ζ-chain expression correlates with expanded Vb clones observed is to be established. The seqeunce of molecular events described in the current work may lead to the Th1 shift observed in aplastic anemia, but further studies are needed to explore the possible role of ζ-chain downregulation to the pathogenesis of AA.

Immune Pathophysiology of Aplastic Anemia

Acquired aplastic anemia (AA) is considered an immune-mediated disease because approximately 70% of AA patients improve with immunosuppressive therapy. However, little is known about the inciting antigens or the mechanisms responsible for the destruction of hematopoietic stem cells by immune system attack. Recent advances in immunologic techniques have promoted our understanding of the pathogenesis of AA and have provided evidence that AA is an organ-specific T-cell-mediated disease localized in the bone marrow. Moreover, antibody screening of patients' serum with a complementary DNA library derived from hematopoietic cells has identified several proteins as candidate autoantigens in AA.

Leukocyte-reduced blood transfusions: perioperative indications, adverse effects, and cost analysis.

Leukocyte-reduced blood transfusions: perioperative indications, adverse effects, and cost analysis.

New Insights into the Pathophysiology of Acquired Cytopenias

This review addresses three related bone marrow failure diseases, the study of which has generated important insights in hematopoiesis, red cell biology, and immune-mediated blood cell injury. In Section I, Dr. Young summarizes the current knowledge of acquired aplastic anemia. In most patients, an autoimmune mechanism has been inferred from positive responses to nontransplant therapies and laboratory data. Cytotoxic T cell attack, with production of type I cytokines, leads to hematopoietic stem cell destruction and ultimately pancytopenia; this underlying mechanism is similar to other human disorders of lymphocyte-mediated, tissue-specific organ destruction (diabetes, multiple sclerosis, uveitis, colitis, etc.). The antigen that incites disease is unknown in aplastic anemia as in other autoimmune diseases; post-hepatitis aplasia is an obvious target for virus discovery. Aplastic anemia can be effectively treated by either stem cell transplantation or immunosuppression. Results of recent trials with antilymphocyte globulins and high dose cyclophosphamide are reviewed. Dr. Abkowitz discusses the diagnosis and clinical approach to patients with acquired pure red cell aplasia, both secondary and idiopathic, in Section II. The pathophysiology of various PRCA syndromes including immunologic inhibition of red cell differentiation, viral infection (especially human parvovirus B19), and myelodysplasia are discussed. An animal model of PRCA (secondary to infection with feline leukemia virus [FeLV], subgroup C) is presented. Understanding the mechanisms by which erythropoiesis is impaired provides for insights into the process of normal red cell differentiation, as well as a rational strategy for patient management. Among the acquired cytopenias paroxysmal nocturnal hemoglobinuria (PNH) is relatively rare; however, it can pose formidable management problems. Since its first recognition as a disease, PNH has been correctly classified as a hemolytic anemia; however, the frequent co-existence of other cytopenias has hinted strongly at a more complex pathogenesis. In Section III, Dr. Luzzatto examines recent progress in this area, with special emphasis on the somatic mutations in the PIG-A gene and resulting phenotypes. Animal models of PNH and the association of PNH with bone marrow failure are also reviewed. Expansion of PNH clones must reflect somatic cell selection, probably as part of an autoimmune process. Outstanding issues in treatment are illustrated through clinical cases of PNH. Biologic inferences from PNH may be relevant to our understanding of more common marrow failure syndromes like myelodysplasia.

New Insights into the Pathophysiology of Acquired Cytopenias

AbstractThis review addresses three related bone marrow failure diseases, the study of which has generated important insights in hematopoiesis, red cell biology, and immune-mediated blood cell injury. In Section I, Dr. Young summarizes the current knowledge of acquired aplastic anemia. In most patients, an autoimmune mechanism has been inferred from positive responses to nontransplant therapies and laboratory data. Cytotoxic T cell attack, with production of type I cytokines, leads to hematopoietic stem cell destruction and ultimately pancytopenia; this underlying mechanism is similar to other human disorders of lymphocyte-mediated, tissue-specific organ destruction (diabetes, multiple sclerosis, uveitis, colitis, etc.). The antigen that incites disease is unknown in aplastic anemia as in other autoimmune diseases; post-hepatitis aplasia is an obvious target for virus discovery. Aplastic anemia can be effectively treated by either stem cell transplantation or immunosuppression. Results of recent trials with antilymphocyte globulins and high dose cyclophosphamide are reviewed.Dr. Abkowitz discusses the diagnosis and clinical approach to patients with acquired pure red cell aplasia, both secondary and idiopathic, in Section II. The pathophysiology of various PRCA syndromes including immunologic inhibition of red cell differentiation, viral infection (especially human parvovirus B19), and myelodysplasia are discussed. An animal model of PRCA (secondary to infection with feline leukemia virus [FeLV], subgroup C) is presented. Understanding the mechanisms by which erythropoiesis is impaired provides for insights into the process of normal red cell differentiation, as well as a rational strategy for patient management.Among the acquired cytopenias paroxysmal nocturnal hemoglobinuria (PNH) is relatively rare; however, it can pose formidable management problems. Since its first recognition as a disease, PNH has been correctly classified as a hemolytic anemia; however, the frequent co-existence of other cytopenias has hinted strongly at a more complex pathogenesis. In Section III, Dr. Luzzatto examines recent progress in this area, with special emphasis on the somatic mutations in the PIG-A gene and resulting phenotypes. Animal models of PNH and the association of PNH with bone marrow failure are also reviewed. Expansion of PNH clones must reflect somatic cell selection, probably as part of an autoimmune process. Outstanding issues in treatment are illustrated through clinical cases of PNH. Biologic inferences from PNH may be relevant to our understanding of more common marrow failure syndromes like myelodysplasia.