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Autoimmune cytopenia is a frequent manifestation of primary immunodeficiencies. Two siblings presented with Evans syndrome, viral infections, and progressive leukopenia. DNA available from one patient showed a homozygous frameshift mutation in tripeptidyl peptidase II (TPP2) abolishing protein expression. TPP2 is a serine exopeptidase involved in extralysosomal peptide degradation. Its deficiency in mice activates cell death programs and premature senescence. Similar to cells from naïve, uninfected TPP2-deficient mice, patient cells showed increased major histocompatibility complex I expression and most CD8+ T-cells had a senescent CCR7-CD127CD28CD57+ phenotype with poor proliferative responses and enhanced staurosporine-induced apoptosis. T-cells showed increased expression of the effector molecules perforin and interferon- with high expression of the transcription factor T-bet. Age-associated B-cells with a CD21 CD11c+ phenotype expressing T-bet were increased in humans and mice, combined with antinuclear antibodies. Moreover, markers of senescence were also present in human and murine TPP2-deficient fibroblasts. Telomere lengths were normal in patient fibroblasts and granulocytes, and low normal in lymphocytes, which were compatible with activation of stress-induced rather than replicative senescence programs. TPP2 deficiency is the first primary immunodeficiency linking premature immunosenescence to severe autoimmunity. Determination of senescent lymphocytes should be part of the diagnostic evaluation of children with refractory multilineage cytopenias.

A specialized form of trogocytosis occurs when Fc receptors on acceptor cells take up and internalize donor cell-associated immune complexes composed of specific monoclonal antibodies (mAbs) bound to target antigens on donor cells. This trogocytosis reaction, an example of antigenic modulation, has been described in recent clinical correlative studies and in vitro investigations for several mAbs used in cancer immunotherapy, including rituximab and ofatumumab. We discuss the impact of Fc-receptor–mediated trogocytosis on the efficacy of cancer immunotherapy and other mAb-based therapies.

Acute myeloid leukemia (AML) in older patients presents a notable therapeutic challenge to the clinical hematologist. The clinical biology of AML among patients is highly heterogeneous. Interpatient variations are relevant for prognosis and treatment choice. Outcome of treatment for patients of advanced age is often compromised by comorbid conditions and an enhanced susceptibility to toxicities from therapy. Here we present selected clinical vignettes that highlight distinct representative situations derived from clinical practice. The vignettes are specifically discussed in light of the perspective of treating older patients with leukemia. We review the clinical significance of various cytogenetic and molecular features of the disease, and we examine the various currently available treatment options as well as the emerging prognostic algorithms that may offer guidance in regard to personalized therapy recommendations. The dilemmas in tailoring treatment selection in this category of patients with AML are the central theme in this discussion.

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by intravascular hemolysis, which is effectively controlled with eculizumab, a humanized monoclonal antibody that binds complement protein 5 (C5). The residual functional activity of C5 can be screened using a 50% hemolytic complement (CH50) assay, which is sensitive to the reduction, absence, and/or inactivity of any components of the classical and terminal complement pathway. Little data exist on complement blockade during treatment. From 2010 to 2012, clinical data, hemolysis biomarkers, complement assessment, and free eculizumab circulating levels were systematically measured immediately before every injection given to 22 patients with hemolytic PNH while receiving eculizumab therapy. During the study, 6 patients received ≥1 red blood cell transfusion. Lack of detectable CH50 activity (defined by CH50 ≤ 10% of normal values) was found in 184 samples (51%) and was significantly associated with lower lactate dehydrogenase levels (P = .002). Low levels of circulating free eculizumab (<50 µg/mL) correlated with detectable CH50 activity (CH50 > 10%; P = .004), elevated bilirubin levels (P < .0001), and the need for transfusions (P = .034). This study suggests that both CH50 activity and circulating free eculizumab levels may help physicians to manage PNH patients receiving eculizumab.

Natural killer (NK) cells can enhance engraftment and mediate graft-versus-leukemia following allogeneic hematopoietic stem cell transplantation (HSCT), but the potency of graft-versus-leukemia mediated by naturally reconstituting NK cells following HSCT is limited. Preclinical studies demonstrate that activation of NK cells using interleukin-15 (IL-15) plus 4-1BBL upregulates activating receptor expression and augments killing capacity. In an effort to amplify the beneficial effects of NK cells post-HSCT, we conducted a first-in-human trial of adoptive transfer of donor-derived IL-15/4-1BBL–activated NK cells (aNK-DLI) following HLA-matched, T-cell–depleted (1-2 x 104 T cells/kg) nonmyeloablative peripheral blood stem cell transplantation in children and young adults with ultra-high-risk solid tumors. aNK-DLI were CD3+-depleted, CD56+-selected lymphocytes, cultured for 9 to 11 days with recombinant human IL-15 plus 4-1BBL+IL-15Rα+ artificial antigen-presenting cells. aNK-DLI demonstrated potent killing capacity and displayed high levels of activating receptor expression. Five of 9 transplant recipients experienced acute graft-versus-host disease (GVHD) following aNK-DLI, with grade 4 GVHD observed in 3 subjects. GVHD was more common in matched unrelated donor vs matched sibling donor recipients and was associated with higher donor CD3 chimerism. Given that the T-cell dose was below the threshold required for GVHD in this setting, we conclude that aNK-DLI contributed to the acute GVHD observed, likely by augmenting underlying T-cell alloreactivity. This trial was registered at www.clinicaltrials.gov as #NCT01287104.

Dyskeratosis congenita (DC) is an inherited multisystem disorder, characterized by oral leukoplakia, nail dystrophy, and abnormal skin pigmentation, as well as high rates of bone marrow (BM) failure, solid tumors, and other medical problems such as osteopenia. DC and telomere biology disorders (collectively referred to as TBD here) are caused by germline mutations in telomere biology genes leading to very short telomeres and limited proliferative potential of hematopoietic stem cells. We found that skeletal stem cells (SSCs) within the BM stromal cell population (BMSCs, also known as BM–derived mesenchymal stem cells), may contribute to the hematologic phenotype. TBD-BMSCs exhibited reduced clonogenicity, spontaneous differentiation into adipocytes and fibrotic cells, and increased senescence in vitro. Upon in vivo transplantation into mice, TBD-BMSCs failed to form bone or support hematopoiesis, unlike normal BMSCs. TERC reduction (a TBD-associated gene) in normal BMSCs by small interfering TERC-RNA (siTERC-RNA) recapitulated the TBD-BMSC phenotype by reducing proliferation and secondary colony-forming efficiency, and by accelerating senescence in vitro. Microarray profiles of control and siTERC-BMSCs showed decreased hematopoietic factors at the messenger RNA level and decreased secretion of factors at the protein level. These findings are consistent with defects in SSCs/BMSCs contributing to BM failure in TBD.

The hematopoietically expressed homeobox gene, Hhex, is a transcription factor that is important for development of definitive hematopoietic stem cells (HSCs) and B cells, and that causes T-cell leukemia when overexpressed. Here, we have used an Hhex inducible knockout mouse model to study the role of Hhex in adult hematopoiesis. We found that loss of Hhex was tolerated in HSCs and myeloid lineages, but resulted in a progressive loss of B lymphocytes in the circulation. This was accompanied by a complete loss of B-cell progenitors in the bone marrow and of transitional B-cell subsets in the spleen. In addition, transplantation and in vitro culture experiments demonstrated an almost complete failure of Hhex-null HSCs to contribute to lymphoid lineages beyond the common lymphoid precursor stage, including T cells, B cells, NK cells, and dendritic cells. Gene expression analysis of Hhex-deleted progenitors demonstrated deregulated expression of a number of cell cycle regulators. Overexpression of one of these, cyclin D1, could rescue the B-cell developmental potential of Hhex-null lymphoid precursors. Thus, Hhex is a key regulator of early lymphoid development, functioning, at least in part, via regulation of the cell cycle.

We examined immunological responses in patients receiving histone deacetylase (HDAC) inhibition (vorinostat) for graft-versus-host disease prophylaxis after allogeneic hematopoietic cell transplant. Vorinostat treatment increased histone acetylation in peripheral blood mononuclear cells (PBMCs) from treated patients, confirming target HDAC inhibition. HDAC inhibition reduced proinflammatory cytokine levels in plasma and from PBMCs and decreased ex vivo responses of PBMCs to proinflammatory TLR-4 stimuli, but did not alter the number or response of conventional T cells to nonspecific stimuli. However, the numbers of regulatory T cells (Tregs) were increased, which revealed greater demethylation of the Foxp3 T regulatory-specific demethylation region. Vorinostat-treated patients showed increased expression of CD45RA and CD31 on Tregs, and these Tregs demonstrated greater suppression on a per cell basis. Consistent with preclinical findings, HDAC inhibition also increased signal transducer and activator of transcription 3 acetylation and induced indoleamine-2,3-dioxygenase. Our data demonstrate that HDAC inhibition reduces inflammatory responses of PBMC but enhances Tregs after allo-HCT.

Patients with t(1;19)-positive acute lymphoblastic leukemia (ALL) are prone to central nervous system (CNS) relapses, and expression of the TAM (Tyro3, Axl, and Mer) receptor Mer is upregulated in these leukemias. We examined the functional role of Mer in the CNS in preclinical models and performed correlative studies in 64 t(1;19)-positive and 93 control pediatric ALL patients. ALL cells were analyzed in coculture with human glioma cells and normal rat astrocytes: CNS coculture caused quiescence and protection from methotrexate toxicity in Merhigh ALL cell lines, which was antagonized by short hairpin RNA–mediated knockdown of Mer. Mer expression was upregulated, prosurvival Akt and mitogen-activated protein kinase signaling were activated, and secretion of the Mer ligand Galectin-3 was stimulated. Merhigh t(1;19) primary cells caused CNS involvement to a larger extent in murine xenografts than in their Merlow counterparts. Leukemic cells from Merhigh xenografts showed enhanced survival in coculture. Treatment of Merhigh patient cells with the Mer-specific inhibitor UNC-569 in vivo delayed leukemia onset, reduced CNS infiltration, and prolonged survival of mice. Finally, a correlation between high Mer expression and CNS positivity upon initial diagnosis was observed in t(1;19) patients. Our data provide evidence that Mer is associated with survival in the CNS in t(1;19)-positive ALL, suggesting a role as a diagnostic marker and therapeutic target.

The acquisition of the cytogenetic abnormalities hyperdiploidy or translocations into the immunoglobulin gene loci are considered as initiating events in the pathogenesis of myeloma and were often assumed to be mutually exclusive. These lesions have clinical significance; hyperdiploidy or the presence of the t(11;14) translocation is associated with a favorable outcome, whereas t(4;14), t(14;16), and t(14;20) are unfavorable. Poor outcomes are magnified when lesions occur in association with other high-risk features, del17p and +1q. Some patients have coexistence of both good and poor prognostic lesions, and there has been no consensus on their risk status. To address this, we have investigated their clinical impact using cases in the Myeloma IX study (ISRCTN68454111) and shown that the coexistence of hyperdiploidy or t(11;14) does not abrogate the poor prognosis associated with adverse molecular lesions, including translocations. We have also used single-cell analysis to study cases with coexistent translocations and hyperdiploidy to determine how these lesions cosegregate within the clonal substructure, and we have demonstrated that hyperdiploidy may precede IGH translocation in a proportion of patients. These findings have important clinical and biological implications, as we conclude patients with coexistence of adverse lesions and hyperdiploidy should be considered high risk and treated accordingly.

The interaction of lymphoid tumor cells with components of the extracellular matrix via integrin αvβ3 allows tumor survival and growth. This integrin was demonstrated to be the membrane receptor for thyroid hormones (THs) in several tissues. We found that THs, acting as soluble integrin αvβ3 ligands, activated growth-related signaling pathways in T-cell lymphomas (TCLs). Specifically, TH-activated αvβ3 integrin signaling promoted TCL proliferation and angiogenesis, in part, via the upregulation of vascular endothelial growth factor (VEGF). Consequently, genetic or pharmacologic inhibition of integrin αvβ3 decreased VEGF production and induced TCL cell death in vitro and in human xenograft models. In sum, we show that integrin αvβ3 transduces prosurvival signals into TCL nuclei, suggesting a novel mechanism for the endocrine modulation of TCL pathophysiology. Targeting this mechanism could constitute an effective and potentially low-toxicity chemotherapy-free treatment of TCL patients.

Mice with T-cell–specific loss of the tumor suppressor gene PTEN early in T-cell ontogeny develop thymic lymphomas that invariably harbor a reciprocal translocation involving the T-cell receptor α/ locus and c-myc, t(14;15). In addition to its known function as a lipid phosphatase opposing PI3K signaling, PTEN has also been described as playing a prominent role in promoting genomic stability. As a result, it has been uncertain which one(s) of these 2 separable features were required to block the development of lymphoma. Here, using a conditional model in which T cells selectively express 1 phosphatase-dead PTEN mutant (C124S) and maintain 1 null allele, we show that PTEN phosphatase activity is required for preventing the emergence of a malignant T-cell population harboring t(14;15), thus constituting a critical function of PTEN in preventing lymphomagenesis.

An unresolved issue in chronic lymphocytic leukemia (CLL) is whether IGHV3-21 gene usage, in general, or the expression of stereotyped B-cell receptor immunoglobulin defining subset #2 (IGHV3-21/IGLV3-21), in particular, determines outcome for IGHV3-21-utilizing cases. We reappraised this issue in 8593 CLL patients of whom 437 (5%) used the IGHV3-21 gene with 254/437 (58%) classified as subset #2. Within subset #2, immunoglobulin heavy variable (IGHV)-mutated cases predominated, whereas non–subset #2/IGHV3-21 was enriched for IGHV-unmutated cases (P = .002). Subset #2 exhibited significantly shorter time-to-first-treatment (TTFT) compared with non–subset #2/IGHV3-21 (22 vs 60 months, P = .001). No such difference was observed between non–subset #2/IGHV3-21 vs the remaining CLL with similar IGHV mutational status. In conclusion, IGHV3-21 CLL should not be axiomatically considered a homogeneous entity with adverse prognosis, given that only subset #2 emerges as uniformly aggressive, contrasting non–subset #2/IGVH3-21 patients whose prognosis depends on IGHV mutational status as the remaining CLL.

Bone marrow megakaryocytes produce platelets by extending long cytoplasmic protrusions, designated proplatelets, into sinusoidal blood vessels. Although microtubules are known to regulate platelet production, the underlying mechanism of proplatelet elongation has yet to be resolved. Here we report that proplatelet formation is a process that can be divided into repetitive phases (extension, pause, and retraction), as revealed by differential interference contrast and fluorescence loss after photoconversion time-lapse microscopy. Furthermore, we show that microtubule sliding drives proplatelet elongation and is dependent on cytoplasmic dynein under static and physiological shear stress by using fluorescence recovery after photobleaching in proplatelets with fluorescence-tagged β1-tubulin. A refined understanding of the specific mechanisms regulating platelet production will yield strategies to treat patients with thrombocythemia or thrombocytopenia.

Inherited thrombocytopenias (ITs) are a heterogeneous group of syndromic and nonsyndromic diseases caused by mutations affecting different genes. Alterations of ACTN1, the gene encoding for α-actinin 1, have recently been identified in a few families as being responsible for a mild form of IT (ACTN1-related thrombocytopenia; ACTN1-RT). To better characterize this disease, we screened ACTN1 in 128 probands and found 10 (8 novel) missense heterozygous variants in 11 families. Combining bioinformatics, segregation, and functional studies, we demonstrated that all but 1 amino acid substitution had deleterious effects. The clinical and laboratory findings of 31 affected individuals confirmed that ACTN1-RT is a mild macrothrombocytopenia with low risk for bleeding. Low reticulated platelet counts and only slightly increased serum thrombopoietin levels indicated that the latest phases of megakaryopoiesis were affected. Given its relatively high frequency in our cohort (4.2%), ACTN1-RT has to be taken into consideration in the differential diagnosis of ITs.

Hemoglobin E (HbE) β-thalassemia is the most common severe thalassemia syndrome across Asia, and millions of people are carriers. Clinical heterogeneity in HbE β-thalassemia is incompletely explained by genotype, and the interaction of phenotypic variation with hepcidin is unknown. The effect of thalassemia carriage on hepcidin is also unknown, but it could be relevant for iron supplementation programs aimed at combating anemia. In 62 of 69 Sri Lankan patients with HbE β-thalassemia with moderate or severe phenotype, hepcidin was suppressed, and overall hepcidin inversely correlated with iron accumulation. On segregating by phenotype, there were no differences in hepcidin, erythropoiesis, or hemoglobin between severe or moderate disease, but multiple linear regression showed that erythropoiesis inversely correlated with hepcidin only in severe phenotypes. In moderate disease, no independent predictors of hepcidin were identifiable; nevertheless, the low hepcidin levels indicate a significant risk for iron overload. In a population survey of Sri Lankan schoolchildren, β-thalassemia (but not HbE) trait was associated with increased erythropoiesis and mildly suppressed hepcidin, suggesting an enhanced propensity to accumulate iron. In summary, the influence of erythropoiesis on hepcidin suppression associates with phenotypic disease variation and pathogenesis in HbE β-thalassemia and indicates that the epidemiology of β-thalassemia trait requires consideration when planning public health iron interventions.

Class IA phosphoinositide 3-kinase β (PI3Kβ) is considered a potential drug target in arterial thrombosis, which is a major cause of death worldwide. Here we show that a striking phenotype of mice with selective p110β deletion in the megakaryocyte lineage is thrombus instability at a high shear rate, which is an effect that is not detected in the absence of p110α in platelets. The high shear rate–dependent thrombus instability in the absence of p110β is observed both ex vivo and in vivo with the formation of platelet emboli. Moreover, PI3Kβ is required for the recruitment of new platelets to a growing thrombus when a pathological high shear is applied. Treatment of human blood with AZD6482, a selective PI3Kβ inhibitor, phenocopies p110β deletion in mouse platelets, which highlights the role of the kinase activity of p110β. Within the growing platelet thrombus, p110β inactivation impairs the activating phosphorylations of Akt and the inhibitory phosphorylation of GSK3. In accord with these data, pharmacologic inhibition of GSK3 restores thrombus stability. Thus, platelet PI3Kβ is not essential for thrombus growth and stability at normal arterial shear but has a specific and critical role in maintaining the integrity of the formed thrombus on elevation of shear rate, suggesting a potential risk of embolization on treatment with PI3Kβ inhibitors.