We searched for JAK2 exon 12 mutations in patients with JAK2 (V617F)-negative myeloproliferative disorders. Seventeen patients with polycythemia vera (PV), including 15 sporadic cases and 2 familial cases, carried deletions or duplications of exon 12 in circulating granulocytes but not in T lymphocytes. Two of the 8 mutations detected were novel, and the most frequent ones were N542-E543del and E543-D544del. Most patients with PV carrying an exon 12 mutation had isolated erythrocytosis at clinical onset, unlike patients with JAK2 (V617F)-positive PV, most of whom had also elevations in white blood cell and/or platelet counts. Both patients with familial PV carrying an exon 12 mutation had an affected sibling with JAK2 (V617F)-positive PV. Thus, several somatic mutations of JAK2 exon 12 can be found in a myeloproliferative disorder that is mainly characterized by erythrocytosis. Moreover, a genetic predisposition to acquisition of different JAK2 mutations is inherited in families with myeloproliferative disorders.

To study the role of the JAK2-V617F mutation in leukemic transformation, we examined 27 patients with myeloproliferative disorders (MPDs) who transformed to acute myeloid leukemia (AML). At MPD diagnosis, JAK2-V617F was detectable in 17 of 27 patients. Surprisingly, only 5 of 17 patients developed JAK2-V617F-positive AML, whereas 9 of 17 patients transformed to JAK2-V617F-negative AML. Microsatellite analysis in a female patient showed that mitotic recombination was not responsible for the transition from JAK2-V617F-positive MPD to JAK2-V617F-negative AML, and clonality determined by the MPP1 polymorphism demonstrated that the granulocytes and leukemic blasts inactivated the same parental X chromosome. In a second patient positive for JAK2-V617F at transformation, but with JAK2-V617F-negative leukemic blasts, we found del(11q) in all cells examined, suggesting a common clonal origin of MPD and AML. We conclude that JAK2-V617F-positive MPD frequently yields JAK2-V617F-negative AML, and transformation of a common JAK2-V617F-negative ancestor represents a possible mechanism. (Blood.

An acquired gain-of-function mutation in the Janus kinase 2 (JAK2-V617F) is frequently found in patients with myeloproliferative disorders (MPDs). To test the hypothesis that JAK2-V617F is the disease-initiating mutation, we examined whether all cells of clonal origin carry the JAK2-V617F mutation. Using allele-specific polymerase chain reaction (PCR) assays for the JAK2 mutation and for the X-chromosomal clonality markers IDS and MPP1, we found that the percentage of granulocytes and platelets with JAK2-V617F was often markedly lower than the percentage of clonal granulocytes determined by IDS or MPP1 clonality assays in female patients. Using deletions of chromosome 20q (del20q) as an autosomal, X-chromosome-independent clonality marker, we found a similar discrepancy IntroductionClonal hematopoiesis has been recognized as a key pathogenetic feature of myeloproliferative disorders (MPDs). 1 Recently, an acquired somatic mutation in the JAK2 gene resulting in a valine-to-phenylalanine substitution at position 617 (JAK2-V617F) was described in patients with MPDs. [2][3][4][5][6] This discovery implied that the presence of the JAK2-V617F mutation could represent the primary causative lesion in MPDs. However, some of the clinical and genetic data suggested that the role of the JAK2-V617F mutation in the MPD pathogenesis could be more complex and led us to propose 2 alternative models for the role of JAK2-V617F in the clonal evolution of MPDs. 5 The first model assumed that JAK2-V617F alone is sufficient to cause MPDs. The second model predicted that MPDs are caused by a mutation in an as-yetunknown gene that precedes the acquisition of the JAK2-V617F mutation and that JAK2-V617F represents a later event in the disease progression. 5 One of the predictions of the first model is that the JAK2-V617F mutation should be present in all cells that represent the MPD clone, whereas according to the second model, only a proportion of clonal cells carry the JAK2-V617F mutation. Here we present data in support of the second model in at least a proportion of patients with MPDs. Cell separations, RNA and DNA isolation, microsatellite PCR All blood samples were processed within 4 hours after collection. Isolation of granulocytes, T lymphocytes, platelets, buccal mucosa, RNA, and DNA, as well as cDNA synthesis, were performed as described. 5,8,9 Loss of heterozygosity on chromosome 20q was determined by microsatellite polymerase chain reaction (PCR) with the markers D20S96 and D20S119 derived from the minimal deleted region. 10 Study design Detection of JAK2-V617FThe allele-specific PCR for JAK2 genotyping was carried out using 20 ng genomic DNA, 45 nM forward primer JAK2-F, and 22.5 nM each of the allele-specific reverse primers JAK2-R-T and JAK2-R-G (Table S1, available at the Blood website; see the Supplemental Materials link at the top of the online article) in a buffer containing 50 mM KCl, 10 mM Tris pH 8.0, and 1.5 mM MgCl 2 . Thirty PCR cycles with denaturing at 94°C for 30 seconds, annealing at 61°C for 30 seconds, and ...

O ver the last decades, incrementally improved xenograft mouse models, supporting the engraftment and development of a human hemato-lymphoid system, have been developed and now represent an important research tool in the field. The most significant contributions made by means of humanized mice are the identification of normal and leukemic hematopoietic stem cells, the characterization of the human hematopoietic hierarchy, and their use as preclinical therapy models for malignant hematopoietic disorders. Successful xenotransplantation depends on three major factors: tolerance by the mouse host, correct spatial location, and appropriately cross-reactive support and interaction factors such as cytokines and major histocompatibility complex molecules. Each of these can be modified. Experimental approaches include the genetic modification of mice to faithfully express human support factors as non-cross-reactive cytokines, to create free niche space, the co-transplantation of human mesenchymal stem cells, the implantation of humanized ossicles or other stroma, and the implantation of human thymic tissue. Besides the source of hematopoietic cells, the conditioning regimen and the route of transplantation also significantly affect human hematopoietic development in vivo. We review here the achievements, most recent developments, and the remaining challenges in the generation of pre-clinicallypredictive systems for human hematology and immunology, closely resembling the human situation in a xenogeneic mouse environment. Humanized hemato-lymphoid system mice

Inhibition of macrophage SIRPα–CD47 interactions mediates phagocytosis and clearance of acute myeloid leukemia stem cells.

The B cell response to lymphocytic choriomeningitis virus is characterized by a CD4 + T cell-dependent polyclonal hypergammaglobulinemia and delayed formation of virus-specific neutralizing antibodies. Here we provide evidence that, paradoxically, because of polyclonal B cell activation, virus-specific T cell help impairs the induction of neutralizing antibody responses. Experimental reduction in CD4 + T cell help in vivo resulted in potent neutralizing antibody responses without impairment of CD8 + T cell activity. These unexpected consequences of polyclonal B cell activation may affect vaccine strategies and the treatment of clinically relevant chronic bacterial, parasitic and viral infections in which hypergammaglobulinemia is regularly found.Lymphocytic choriomeningitis virus (LCMV) is a noncytopathic RNA virus that is initially controlled by a strong CD8 + T cell response in a perforin-dependent way 1,2 . In contrast, LCMV-neutralizing antibodies usually develop late in infection, after day 50, and remain at low titer 3 . This phenomenon is also found after infection with other poorly cytopathic or noncytopathic viruses such as human immunodeficiency virus (HIV) 4 and hepatitis C virus 5,6 . Neutralizing antibodies directed against low-cytopathic viruses increase in titer when CD8 + T cell (cytotoxic T lymphocyte (CTL)) function is impaired or absent 3,7,8 . Possible mechanisms have been postulated to include CD8 + T cell-dependent immunopathology 9,10 , enhanced viral replication in the absence of CTLs 2,8 or the need for affinity or avidity maturation of specific antibodies. These observations also suggest a competitive relationship between cellular and humoral antiviral immune responses, as has been successfully modeled theoretically 11 . LCMV infection induces a strong CD4 + T cell-dependent, virusnonspecific, polyclonal hypergammaglobulinemia 12,13 , a phenomenon often associated with 'persistence-prone' , low-cytopathic viruses like HIV and hepatitis C virus or with chronic bacterial and parasitic infections. Yet a biological function for polyclonal B cell activation has remained elusive 14 .Here we have evaluated the function of T helper cells and found that paradoxically, because of polyclonal B cell activation, CD4 + T cells impaired the formation of virus-neutralizing antibodies and favored virus persistence. This inverse causal relationship of T cell help and virus-specific antibody formation was modified only indirectly by CD8 + T cells. Experimental deliberate suppression of T helper responses after LCMV infection resulted in enhanced neutralizing antibody formation while leaving CTL responses intact. RESULTS Polyclonal B cell activation versus neutralizing antibodiesCompared with results obtained with control C57BL/6 mice, infection of CD8 + T cell-depleted C57BL/6 or CD8a-deficient (Cd8a À/À ) mice with high-dose (2 Â 10 6 PFU) LCMV, strain WE (LCMV-WE) resulted in increased titers of neutralizing antibodies (Fig. 1a), correlating with increased viral replication (Fig. 1b). However, virusindu...

SummaryData on angiogenesis in the bone marrow of BCR‐ABL1‐negative myeloproliferative neoplasm (MPN) patients suggest an increase of the microvessel density (MVD) and vascular endothelial growth factor (VEGF) expression, but relations to the JAK2‐V617F status remain controversial. We performed immunohistochemical studies of MVD and VEGF‐expression in 100 MPN, including 24 essential thrombocythemia‐ (ET), 46 polycythemia vera‐ (PV), 26 primary myelofibrosis‐ (PMF), four myelodysplastic (MDS)/MPN‐ and 20 control reactive bone marrow cases, and correlated these findings with biological and clinical key data and the JAK2‐V617F status. We found significantly increased MVD, particularly that assessed by CD105, and VEGF expression in MPN compared to controls (PMF > PV > MDS/MPN > ET). We observed stronger association between CD105‐MVD and VEGF expression, fibrosis, and JAK2‐V617F mutant allele burden, compared to CD34‐MVD. MVD was strongly increased in MPN with high JAK2‐V617F mutant allele burden. Our study highlights the importance of newly formed CD105+ vessels in the bone marrow of MPN patients, and indicates that assessment of CD105‐MVD better reflects angiogenic activity in MPN. In addition, it provides evidence that despite the fact that angiogenesis is generally independent of the JAK2‐V617F status in MPN, new vessel formation might be linked to Jak2 effects in some cases with high JAK2‐V617F mutant allele burden.

We studied the lineage distribution of JAK2 mutations in peripheral blood of 8 polycythemia vera (PV) patients with exon 12 mutations and in 21 PV patients with JAK2-V617F. Using a quantitative allele discrimination assay, we detected exon 12 mutations in purified granulocytes, monocytes, and platelets of 8 patients studied, but lymphoid cells showed variable involvement and the mutation was absent in T cells. Endogenous erythroid colonies grew in all patients analyzed. One patient displayed erythroid colonies homozygous for the exon 12 mutation with evidence for mitotic recombination on chromosome 9p. In some patients with exon 12 mutations or JAK2-V617F, a proportion of endogenous erythroid colonies were negative for both JAK2 mutations. One patient carried 2 independent clones: one with an exon 12 mutation and a second with JAK2-V617F. IntroductionMutations in exon 12 of JAK2 are detected selectively in patients with polycythemia vera (PV) that are negative for JAK2-V617F and in some patients with idiopathic erythrocytosis. 1 The JAK2-V617F and exon 12 mutations represent clonal markers useful to track the hematopoietic lineages involved in myeloproliferative disorder (MPD). [2][3][4][5][6] In patients with MPD, JAK2-V617F is present in purified hematopoietic stem cells, in myeloid lineages of the peripheral blood, and in variable proportions of lymphoid cells. [7][8][9][10][11] Using a novel sensitive assay, we quantitated the involvement of exon 12 mutations in purified peripheral blood lineages and in erythroid progenitor assays. In addition, we addressed the question of whether JAK2-V617F is present in T cells by clonal analysis. Methods PatientsThe screening for JAK2 exon 12 mutation in MPD patients was performed by DNA sequencing. 12 All patients except p024 fulfilled the diagnostic criteria of PV according to the World Health Organization (Table S1, available on the Blood website; see the Supplemental Materials link at the top of the online article). 13,14 Patient p024 was initially diagnosed with essential thrombocythemia, and several months later phlebotomies were started because of rising hemoglobin (175 g/L). Two patients with JAK2 exon 12 mutations (Vi064, Vi327) were from Vienna, Austria. All other patients were from Basel, Switzerland. The collection of patient samples was approved by the "Ethik Kommission Beider Basel" and the "Ethik Kommission der Universität Wien und des Allgemeinen Krankenhauses der Stadt Wien-AK." Written consent was obtained from all patients in accordance with the Declaration of Helsinki. Cells, DNA, and RNA analysesIsolation of granulocytes, platelets, and peripheral blood mononuclear cells was performed as described. 5,15 Sorting of peripheral blood mononuclear cells, colony assays in methylcellulose, T-cell cloning, 16 and the SNaPshot assay for RNA samples are described in Document S1. The allele discrimination assay for detection and quantification of JAK2 exon 12 mutations is described in Figure S1. Allele-specific polymerase chain reaction (PCR) for the detection ...