Persistent imbalance, anti-apoptotic, and anti-inflammatory signature of circulating C-C chemokines and cytokines in patients with paroxysmal nocturnal hemoglobinuria.

OBJECTIVE: Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal non-malignant disease in which hematopoietic cell apoptosis may play an important pathophysiological role. Previous studies of the content of phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3) indicated the possibility of remote transmission of anti-apoptotic signals between pathological and normal hematopoietic progenitors. METHODS: The study determined the plasma levels of beta chemokines and cytokines in N = 19 patients with PNH and 31 healthy controls. The research material was peripheral blood plasma (EDTA) stored at -80 °C until the test. Beta chemokine and cytokine concentrations were tested in duplicate with Bio-Plex Pro Human Cytokine Assay (Bio-Rad, Hercules, CA, USA) using a Luminex 200 flow cytometer and xPONENT software (Luminex Corporation, Austin, TX, USA). In peripheral blood CD34+ cells we tested the proportions of PI(3,4,5)P3+ and Annexin binding apoptotic phenotype using FC and phosflow. RESULTS: Compared to the control group, the PNH group showed a significant increase in the plasma concentration of some beta chemokines and cytokines, including MIP-1alpha/CCL3, eotaxin/CCL11, MCP1/CCL2, IL4 and G-CSF. In the group of PNH patients, a significant decrease in the concentration of some cytokines was also observed: RANTES/CCL5, MIP-1beta/CCL4, PDGF-BB and IL9. At the same time, the plasma concentrations of the chemokine IP-10/CXCL10 and the cytokines IFN-gamma, TNF, IL6 and IL10 showed no significant deviations from the values for the control group. Anti-apoptotic phenotype and phosphatidylinositol (3,4,5)-trisphosphate content in PNH clone of CD34+ cells were associated with the level of CCL3 and negatively associated with CCL5, CCL4, PDGF-BB and IL9. CONCLUSIONS: This data suggest the existence of apoptotic and PI(3,4,5)P3 imbalance in PNH CD34+ cells driven by anti-apoptotic cytokine biosignature in PNH. Plasma cytokines and intracellular enzymes that regulate the phosphoinositide pathways may become a therapeutic target in PNH.

Allogeneic Hematopoietic Stem Cell Transplantation for Paroxysmal Nocturnal Hemoglobinuria: Multicenter Analysis by the Polish Adult Leukemia Group.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the sole potential cure for paroxysmal nocturnal hemoglobinuria (PNH); however, the data on its utility in PNH are limited. This retrospective analysis of patients with PNH who underwent allo-HSCT in 11 Polish centers between 2002 and 2016 comprised 78 patients with PHN, including 27 with classic PNH (cPNH) and 51 with bone marrow failure-associated PNH (BMF/PNH). The cohort was 59% male, with a median age of 29 years (range, 12 to 65 years). There was a history of thrombosis in 12% and a history of hemolysis in 81%, and 92% required erythrocyte transfusions before undergoing allo-HSCT. No patient received eculizumab, and 26% received immunosuppressive treatment. The median time from diagnosis to allo-HSCT was 12 months (range, 1 to 127 months). Almost all patients (94%) received reduced-toxicity conditioning, 66% with treosulfan. The stem cell source was peripheral blood in 72% and an identical sibling donor in 24%. Engraftment occurred in 96% of the patients. With a median follow-up of 5.1 years in patients with cPNH and 3.2 years in patients with BMF/PNH, 3-year overall survival (OS) was 88.9% in the former and 85.1% in the latter (P = not significant [NS]). The 3-year OS for patients with/without thrombosis was 50%/92% (P = NS) in the cPNH group and 83.3%/85.3% (P = NS) in the BMF/PNH group. The 3-year OS for in the BMF/PNH patients with/without hemolysis was 93.9%/62.9% (hazard ratio, .13; P = .016). No other factors impacted OS. After allo-HSCT, the frequency of the PNH clone was reduced to 0%, <1%, and <2.4% in 48%, 48%, and 4% of cPNH patients and in 84%, 11%, and 5% of BMF/PNH patients, respectively. The frequency of acute graft-versus-host disease (GVHD) grade II-IV was 23%, and the cumulative 1-year incidence of extensive chronic GVHD was 10.8% in the BMF/PNH group and 3.7% in the cPNH group. Allo-HSCT is a valid option for PNH patients, effectively eliminating the PNH clone with satisfactory overall survival and acceptable toxicity. Reduced-toxicity conditioning with treosulfan is effective and safe in patients with cPNH and BMF/PNH.

Further evidence that paroxysmal nocturnal haemoglobinuria is a disorder of defective cell membrane lipid rafts.

The glycolipid glycosylphosphatidylinositol anchor (GPI-A) plays an important role in lipid raft formation, which is required for proper expression on the cell surface of two inhibitors of the complement cascade, CD55 and CD59. The absence of these markers from the surface of blood cells, including erythrocytes, makes the cells susceptible to complement lysis, as seen in patients suffering from paroxysmal nocturnal haemoglobinuria (PNH). However, the explanation for why PNH-affected hematopoietic stem/progenitor cells (HSPCs) expand over time in BM is still unclear. Here, we propose an explanation for this phenomenon and provide evidence that a defect in lipid raft formation in HSPCs leads to defective CXCR4- and VLA-4-mediated retention of these cells in BM. In support of this possibility, BM-isolated CD34(+) cells from PNH patients show a defect in the incorporation of CXCR4 and VLA-4 into membrane lipid rafts, respond weakly to SDF-1 stimulation, and show defective adhesion to fibronectin. Similar data were obtained with the GPI-A(-) Jurkat cell line. Moreover, we also report that chimeric mice transplanted with CD55(-/-)  CD59(-/-) BM cells but with proper GPI-A expression do not expand over time in transplanted hosts. On the basis of these findings, we propose that a defect in lipid raft formation in PNH-mutated HSPCs makes these cells more mobile, so that they expand and out-compete normal HSPCs from their BM niches over time.

Potential link between MHC-self-peptide presentation and hematopoiesis; the analysis of HLA-DR expression in CD34-positive cells and self-peptide presentation repertoires of MHC molecules associated with paroxysmal nocturnal hemoglobinuria.

The mechanisms of MHC allele associations with paroxysmal nocturnal hemoglobinuria (PNH) and its aplastic anemia subtype (AA/PNH) remain unclear. It might be dependent on MHC molecule functional properties, such as a scope and frequency of antigen sampling and presentation. For documented PNH-associated MHC alleles we analyzed current reference databases on MHC molecule-eluted peptide presentation repertoires and searched for a range of presented peptides. MHC class II expression was measured on CD34+ cells and appeared to be increased in PNH patients. Two class I alleles (HLA-A*24:02 and B*18:01) have been previously confirmed to associate with protection and increased risk of AA/PNH, respectively. Their product molecules presented immunodominant epitopes derived from proapoptotic (serine/threonine-protein phosphatase) and antiapoptotic (phospholipase D), respectively, intracellular enzymes dependent on phosphoinositide (PI) content. For total PNH and non-aplastic PNH (n/PNH) subtype-associated DRB1*15:01 and DRB1*04:01 class II molecules presentation of exceptionally broad arrays of their own peptide fragments has been found. We conclude that self antigen peptides presented with high frequency in the context of MHC molecules of increased expression may be involved in the immune recognition and the regulation of HSC in the periphery. The block in the normal plasma membrane PI production due to the PIG-A mutation can help explain the differences in the activation of intracellular regulatory pathways observed between PNH and normal HSC. This is evident in the variation in MHC association patterns and peptide presentation repertoires between these two groups of patients.

The patterns of MHC association in aplastic and non-aplastic paroxysmal nocturnal hemoglobinuria.

The deficiency of glycosyl-phosphatidylinositol (GPI)-anchored proteins in plasma membranes of PIG-A gene mutated hematopoietic stem cells (HSCs) is so far insufficient to explain the domination of paroxysmal nocturnal hemoglobinuria (PNH) clone over the normal HSC. We attempted to elucidate possible link between MHC and initial severe aplastic anemia (ISAA/PNH) type and non-aplastic (n/PNH) outcome of PNH. In 50 PNH patients assigned as ISAA/PNH (n = 13), n/PNH (n = 33) or nonassigned (n = 4) and 200 ethnically matched controls we analyzed MHC associations. Our data confirmed strong associations of DRB1*15:01 (RR = 3.51, p = 0.0011) and DQB1*06:02 (RR = 7.09, p = 0.000026) alleles, especially with n/PNH subtype. B*18:01 allele was associated with increased risk of ISAA/PNH subtype (RR = 5.25, p = 0.0028). We conclude that both class II and class I MHC alleles are associated with different subsets of PNH. Clonal selection of PIG-A mutated cells with cognate metabolic block is associated with MHC class II alleles DRB1*15:01 and DQB1*06:02 independent from initial severe AA clone selection. MHC class I molecule B*18:01 can additionally influence the domination of PNH clone in PNH subjects with initial severe aplastic anemia.

First two successful unrelated donor bone marrow transplantations for paroxysmal nocturnal hemoglobinuria in Poland.

OBJECTIVES: The experience with bone marrow transplantation (BMT) from matched unrelated donors (MUD) for paroxysmal nocturnal hemoglobinuria (PNH) is limited and optimal preparative regimen has been not established. METHODS: We report first two MUD BMTs for patients with PNH in Poland. Preparative regimen consisted of Treosulfan, Fludarabine and Thymoglobulin. We also present the review of published reports on allogeneic transplantations for PNH and discuss important transplant-related issues. RESULTS: Both patients are alive and are doing well over 12 and over 4 months following BMT. Regeneration is complete with full 100% donor chimerism and the eradication of PNH clone. CONCLUSIONS: MUD BMT is an effective treatment for PNH. Treosulfan, Fludarabine and Thymoglobulin treatment can be safely and effectively used for conditioning in PNH.