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Summary 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 Vβ2 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.

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.

Fanconi anaemia (FA) is a rare genetic disorder that predisposes to the development of aplastic anaemia and neoplasia. The pathophysiologic hallmark of FA is increased susceptibility to chromosomal breakage. Superoxide metabolism has also been shown to be involved in the cellular pathophysiology of FA. Human SOD (rh-SOD), an enzyme which dismutates superoxide, has recently been cloned and expressed in yeast. We treated four FA patients with a 2-week infusion of rh-SOD (25 mg/kg d daily) to determine whether rh-SOD had any effect on haemopoietic progenitor cell growth or on the abnormal cellular phenotype. We found that lymphocyte chromosomal aberrations induced by diepoxybutane were decreased during rh-SOD treatment in two patients and that bone marrow progenitors were increased in one patient.

Abstract 1317 Poster Board I-333 Thrombocytopenia occurs in aplastic anemia (AA) as a result of hematopoietic failure, in which decreased or absent megakaryocytes are observed in a hypocellular bone marrow. In immune thrombocytopenia (ITP), thrombocytopenia primarily occurs secondary to peripheral destruction and marrow cellularity is normal with megakaryocytes numerically normal or increased. In both AA and ITP, platelet function is normal and the disturbance in hemostasis is the result of the diminution in the platelet count. We recently reported that high plasma concentrations of Tpo and G-CSF, in combination with low levels of CD40L, CXCL5, CCL5, CXCL11, EGF, VEGF, and eotaxin, served as a cytokine signature profile for AA when compared to MDS or healthy controls (Blood 2008;112:380-381). In 8 patients with severe AA who responded to immunosuppressive therapy, the levels of CD40L, CXCL5, EGF, and CCL5 in plasma correlated with platelet recovery, as compared with absolute neutrophil count and hemoglobin recovery. We hypothesized that the plasma levels of CD40L, CXCL5, EGF, and CCL5 reflected platelets or their precursor megakaryocytes. We measured these cytokines in the plasma of 41 ITP patients using the Luminex assay, for comparison with 33 untreated AA patients and 52 healthy controls (Figure 1). Results were assessed by the Kruskal-Willis one-way analysis of variance statistic. ITP patients showed similar low levels of circulating CCL5, CD40L, CXCL5, and EGF as did AA patients, when compared to healthy controls. These low plasma levels of cytokines/chemokines in AA and ITP may not reflect inflammation but rather the common feature of deficiency of platelets. Soluble CD40L appears to be a platelet agonist and may promote coagulation; CCL5-deficient mice model demonstrate that CCL5 blockade compromises systemic immune responses, delays macrophage-mediated viral clearance and impairs normal T cell function; CXCL5 attracts leukocytes and activates platelets; and EGF upregulates tissue factor and acts as a procoagulant. Levels of Tpo, G-CSF, VEGF, and eotaxin were not statistically different between ITP and healthy controls, but the levels of these cytokines were different between AA patients and healthy controls, indicating that Tpo levels are high when thrombocytopenia is due to megakaryocyte deficiency and low when due to increased platelet destruction. High Tpo, and G-CSF levels in AA represent a compensatory physiologic mechanism operating to counter impaired blood hematopoiesis. In addition, ITP patients showed higher levels of IP-10 than both AA patients and healthy controls, suggesting the involvement of interferon-gamma related responses in ITP. We compared cytokine gene expression in platelets, differentiated CD61+ cells (megakaryocytes), and CD61- cells (non-megakaryocytes) from bone marrow CD34+ cells of healthy controls by real-time PCR. Platelets and megakaryocytes showed higher mRNA expression of CXCL5 (440 and 16.3-fold), CD40L (9.6 and 51.9-fold), EGF (171 and 112-fold) and CCL5 (526 and 27.7-fold) than did CD61- cells (non-megakaryocytes), respectively. Together, our study provides evidence that platelets as well as megakaryocytes appear to be sources of CXCL5, CCL5, EGF, and CD40L; these cell types may contribute to the physiological levels of these cytokines. Similarities in cytokine levels between AA and ITP may reflect the autoimmune destruction of targets at varying stages of differentiation. Measurement of platelet-associated cytokines allows inferences concerning pathophysiology in quantitative platelet disorders. Figure 1 Comparison of plasma cytokine levels among patients with immune thrombocytopenia (ITP), aplastic anemia (AA), and healthy controls (HC). Cytokine levels in the plasma samples from 41 ITP, 33 untreated AA patients and 52 HC were measured using Luminex assay. The bars represent median values. ***, p < .001; *, p < .05; ns, not significant (Kruskall-Wallis). Tpo, Thrombopoietin; G-CSF, granulocyte colony-stimulating factor; IP-10, Interferon-inducible protein-10; CXCL5, chemokine (C-X-C motif) ligand 5; CCL5, chemokine (C-C motif) ligand 5; CD40L, CD40 ligand; EGF, epidermal growth factor; VEGF, vascular endothelial growth factor. Figure 1. Comparison of plasma cytokine levels among patients with immune thrombocytopenia (ITP), aplastic anemia (AA), and healthy controls (HC). Cytokine levels in the plasma samples from 41 ITP, 33 untreated AA patients and 52 HC were measured using Luminex assay. The bars represent median values. ***, p < .001; *, p < .05; ns, not significant (Kruskall-Wallis). Tpo, Thrombopoietin; G-CSF, granulocyte colony-stimulating factor; IP-10, Interferon-inducible protein-10; CXCL5, chemokine (C-X-C motif) ligand 5; CCL5, chemokine (C-C motif) ligand 5; CD40L, CD40 ligand; EGF, epidermal growth factor; VEGF, vascular endothelial growth factor. Disclosures No relevant conflicts of interest to declare.

Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal stem cell disorder characterized by intravascular hemolysis, thrombosis, and bone marrow failure; in PNH, mutations in the PIG-A gene prevent synthesis of glycosylphosphoinositol (GPI) anchors and therefore cell membrane expression of GPI-anchored proteins. PNH cell clonal expansion occurs in the setting of immune-mediated hematopoietic cell destruction. The mechanism responsible for PNH clonal expansion is uncertain, but there is experimental evidence for relative resistance of GPI-anchored protein negative cells to NK-cell mediated cytotoxicity. Signaling lymphocyte activation molecule (SLAM; CD150; SLAMF1) is a glycosylated type-I transmembrane protein of relative molecular mass 70 Kd; the protein has a cytoplasmic domain bearing a paired immunoreceptor tyrosine-based switch motif. SLAM is constitutively expressed on the surface of activated T-, B- lymphocytes, dendritic cells and macrophages with a high-affinity self ligand. SLAM functions include co-stimulation of T and B cells, augmentation of T-cell cytotoxicity, and regulation of proliferation and differentiation of B cells. The recruitment of SLAM or SLAM-associated proteins is important in modulating innate and adaptive immune responses. Engagement of CD244 (2B4), another SLAM family member, by CD48--a GPI-anchored protein--results in CD244 activation, leading to NK cell stimulation and NK cell mediated cytotoxicity. We examined SLAM and CD244 expression in peripheral blood mononuclear cells from patients with PNH and healthy controls to establish their involvement in PNH. We first analyzed SLAM expression by flow cytometry on CD56+ and CD3+ cells from 11 PNH patients (5 females, 6 males) and 10 healthy controls. In CD56+ cells membrane SLAM (mSLAM) was up-regulated in 10 of 11 patients compared to controls (p=0.04). mSLAM was more modestly elevated in CD3+ cells from patients (p=0.04). Expression of CD244 also showed increased expression in PNH patients’ CD56+ cells (p=0.0008) and in CD3+ (p= 0.05). mRNA levels, measured by RT-PCR, of mSLAM in patients (n=4) and controls (n=4) were consistent with flow cytometric data, PNH patients having increased mSLAM mRNA levels (p=0.18). In a single PNH patient who showed up-regulation by flow and RT-PCR we also detected increased mSLAM protein levels by immunoblot. RT-PCR was also employed to quantitate mRNA levels of soluble or cytoplasmic SLAM (sSLAM): sSLAM expression was decreased in patients (n=4) compared to controls (n=3) (p< 0.0001). All differences between groups were evaluated using the F test to compare variances. In summary, NK cells from PNH patients have increased levels of mSLAM and 2B4; up-regulation suggests involvement of SLAM family members in immunologically mediated selection of PNH clones in immune-mediated marrow failure syndromes and is consistent with a role for NK cells in PNH clonal expansion.

Aplastic anemia is a bone-marrow-failure syndrome characterized by low blood-cell counts and a fatty bone marrow. In most cases, no obvious etiological factor can be identified, but clinical responses to immunosuppressive treatment (IST) strongly suggest an immune pathophysiology. Our previous study of T-cell receptor (TCR) Vβ (variable region of β-chain) repertoire usage by flow cytometry suggested that aplastic anemia results from antigen-specific lymphocyte attack on hematopoietic progenitors (Risitano et al. Lancet2004; 364:355). In the current work, 7 patients were investigated for Vβ pattern expression before first immunosuppresive treatment, at the remission, and again on relapse. The TCR Vβ repertoire was analyzed for CD4+ and CD8+ subsets, separately, by flow cytometry, using a monoclonal antibody set of 22 different Vβ chains. Most patients had very different patterns of Vβ usage from healthy individuals, and all but one showed expansion of at least one Vβ family before immunosuppressive treatment (Vβ family expansions were defined as 2 standard deviations (SD) from the means in controls). The median number of expanded Vβ families was 4 per patient among CD8CD28dim effector cells. At remission, almost all the initially expanded Vβ subfamilies decreased to less than 2SD of controls. At relapse, most of the expanded Vβ subsets were increased again. However, 5/7 patients showed new expanded Vβ subsets at recurrence of cytopenias, suggesting antigenic spread of new epitopes recognized by immune systems. Although no common pattern of specific expanded Vβ subsets could be identified among different patients, some Vβ subfamilies appeared to be more frequently involved (Vβ 5.1 and Vβ 5.2 were expanded in 4 of 7 patients both at initial presentation and relapse ). These data suggest that monitoring Vβ subsets in aplastic anemia, and potentially in other immune-mediated human diseases of a similar pathophysiology could be used to guide individual therapeutic decisions and in the development of new treatments.

Cell number, protein, and phorbol myristate acetate (PMA)-induced H2O2 production were measured in cultured human peripheral blood monocytes for six days after exposure to varying doses of gamma-radiation. Both the number of adherent cells and the protein per dish decreased with increasing radiation doses. The dose of radiation decreasing the number of adherent cells by 37% on days 4 and 6 postirradiation was 29 Gy. Four hours postirradiation there was a small decrease in PMA-induced H2O2 production for doses of 7.5 Gy or greater; levels returned to normal by eight hours and increased at 24 hours postirradiation. By day 4 postirradiation significant increases in PMA-induced H2O2 production were noted at all radiation doses (2.5 to 50 Gy). This increase was not due to a shift in the PMA dose-response curve, a change in the time course of the PMA response, or an effect of decreased cell density on the assay system. Superoxide levels were not significantly changed in cells exposed to 20 Gy. Catalase, glutathione peroxidase, and superoxide dismutase levels also were unchanged. Culturing irradiated cells with gamma-interferon increased PMA-induced H2O2 release, which indicated that irradiated cells retained their capacity to respond to gamma-interferon. These data demonstrate that irradiation affects the PMA-induced H2O2 production of human monocytes in a time- and dose- dependent manner. An increase in the release of reactive oxygen intermediates by the macrophage may play a role in enhancing the deleterious effects of radiation in vivo.