Bone Marrow Eosinophilia Research Articles

A rare case of PDGFRA-positive chronic eosinophilic leukemia.

Chronic eosinophilic leukemia (CEL) is a clonal proliferation of eosinophilic precursors resulting in a persistently elevated number of eosinophils in blood, bone marrow, and peripheral tissues. The vaguely overlapping clinicopathological picture of CEL with idiopathic hypereosinophilic syndrome (IHES) often adds to the diagnostic dilemma. Here, we report a rare case of 49-year-old male who presented with multiorgan damage. Peripheral blood smear revealed predominantly eosinophils with dysplastic changes, shift to left in eosinophilic series, and 2% blasts. The bone marrow showed 5% blasts and many eosinophils, precursors of eosinophils, and myeloid and megakaryocytic proliferation. Till date, CEL is a very rare entity. Its presentation with cardiac and neurological manifestations is still rare.

Benralizumab for allergic asthma: a randomised, double-blind, placebo-controlled trial.

Benralizumab induces rapid and near-complete depletion of eosinophils from blood and lung tissue. We investigated whether benralizumab could attenuate the allergen-induced late asthmatic response (LAR) in participants with allergic asthma. Participants with allergic asthma who demonstrated increased sputum eosinophils and LAR at screening were randomised to benralizumab 30 mg or matched placebo given every 4 weeks for 8 weeks (3 doses). Allergen challenges were performed at weeks 9 and 12 when blood, sputum, bone marrow and bronchial tissue eosinophils and LAR were assessed. 46 participants (mean age 30.9 years) were randomised to benralizumab (n=23) or placebo (n=23). Eosinophils were significantly reduced in the benralizumab group compared with placebo in blood at 4 weeks and sputum and bone marrow at 9 weeks after treatment initiation. At 7 h after an allergen challenge at week 9, sputum eosinophilia was significantly attenuated in the benralizumab group compared to placebo (least squares mean difference -5.81%, 95% CI -10.69- -0.94%; p=0.021); however, the LAR was not significantly different (least squares mean difference 2.54%, 95% CI 3.05-8.12%; p=0.363). Adverse events were reported for seven (30.4%) and 14 (60.9%) participants in the benralizumab and placebo groups, respectively. Benralizumab administration over 8 weeks resulted in a significant attenuation of blood, bone marrow and sputum eosinophilia in participants with mild allergic asthma; however, there was no change in the LAR, suggesting that eosinophils alone are not a key component of allergen-induced bronchoconstriction.

Interactions between eosinophils and IL-5Rα–positive mast cells in nonadvanced systemic mastocytosis

Bidirectional interactions between eosinophils and mast cells (MCs) have been reported in various allergic diseases. Bone marrow (BM) eosinophilia, and to a lesser extent blood eosinophilia, is common in systemic mastocytosis (SM), but its significance remains unknown. To describe blood and BM eosinophil characteristics in SM. A large collection of BM biopsies was analyzed using immunohistochemical staining and whole-slide imaging. Eosinophil and extracellular granules were detected by eosinophil peroxidase (EPX) staining, and MCs by KIT staining. Complementary analyses were conducted using flow cytometry and immunofluorescence. Eosinophil infiltrates and large areas of eosinophil degranulation were observed within or around BM MC infiltrates in SM. EPX staining surface, highlighting intact eosinophils and eosinophil degranulation, was higher in non-advanced-SM (n=37 BM biopsies) compared to both controls (n=8, p=0.0003) and to advanced SM (n=24, p=0.014). In non-advanced SM, positive correlations were observed between serum tryptase levels and percentages of eosinophil counts in BM aspirations (Spearman r coefficient r=0.38, p=0.038), eosinophils count in BM biopsies (r=0.45, p=0.007), EPX staining (r=0.37, p=0.035) and eosinophil degranulation (r=0.39, p=0.023). Eosinophil counts in BM biopsies also correlated with MC counts (r=0.47, p=0.006) and KIT staining surface (r=0.49, p=0.003). BM MCs expressed interleukin-5 receptor and other usual eosinophil cytokine/chemokine receptors, and blood eosinophils display several increased surface markers compared to controls, suggesting an activated state. Our data suggest a possible crosstalk between MCs and eosinophils, supporting MC tryptase release and MC activation-related symptoms. This suggests a rationale for targeting eosinophils in non-advanced-SM not fully controlled by other therapies.

Value and limitations of targeted next-generation sequencing in idiopathic hypereosinophilia: an integrative diagnostic tool in challenging cases

Here, we reviewed clinical-morphological data and investigated mutational profiles by NGS in a single-center series of 28 consecutive patients admitted to our hospital between September 2011 and November 2021 for idiopathic hypereosinophilia (HE).Bone marrow (BM) morphology was evaluated in 22 patients: while in six subjects BM was unremarkable, in the remaining cases an increase in BM eosinophils was observed, together with a slight increase in BM fibrosis (MF-1) in 5/22 patients.A total of 4/28 patients had at least one genetic lesion by targeted NGS. In particular, the genes involved were: two each of TET2 and DNMT3A; and one each of JAK2V617F, ASXL1, PPM1D, and ZBTB33. Notably, JAK2V617F and TET2 mutations co-occurred, with the JAK2V617F-mutated sample also carrying TET2 lesions. Median VAF was 21%, with the exception of the oncodriver JAK2V617F, which showed a VAF > 50% in the reported case. Of note, of the four cases bearing lesions, 2/4 had multiple hits in different genes.While in recent years mutational analysis using NGS has proven to be able to differentiate clonal hematopoietic neoplasms from reactive processes in diagnostically difficult cases, we found somatic mutations in only 14.3% of patients who acceded to our hospital for idiopathic HE. More importantly, excluding the JAK2V617F-mutated case with an underlying MPN-Eo diagnosis, NGS was able to identify somatic mutations in only three cases, all older than 70 years. Consequently, the detection of these mutations in idiopathic HE patients should be interpreted with caution and only in the context of other supportive clinical-pathological findings.

Flow cytometric immunophenotypic differentiation patterns of bone marrow eosinophilopoiesis.

Flow cytometry has been widely used to study immunophenotypic patterns of maturation of most hematopoietic lineages in normal human bone marrow aspirates, thus allowing identification of changes in patterns in many myeloid malignancies. Eosinophils play an important role in a wide variety of disorders, including some myeloid neoplasms. However, changes in flow cytometric immunophenotypic patterns during normal and abnormal bone marrow eosinophilopoiesis have not been well studied. Fresh bone marrow aspirates from 15 healthy donors, 19 patients with hypereosinophilic syndromes (HES), and 11 patients with systemic mastocytosis (SM) were analyzed for candidate markers that included EMR-1, Siglec-8, CCR3, CD9, CD11a, CD11b, CD11c, CD13, CD16, CD29, CD34, CD38, CD45, CD44, CD49d, CD49f, CD54, CD62L, CD69, CD117, CD125 (IL-5Rα), HLA-DR, using 10 parameter flow cytometry. Putative CD34-negative immature and mature normal eosinophil populations were first identified based on changes in expression of the above markers in healthy donors, then confirmed using fluorescence-based cell sorting and morphological evaluation of cytospin preparations. The normal immunophenotypic patterns were then compared to immunophenotypic patterns of eosinophilopoiesis in patients with HES and SM. The eosinophilic lineage was first verified using the human eosinophil-specific antibody EMR-1 in combination with anti-IL-5Rα antibody. Then, a combination of Siglec-8, CD9, CD11b, CCR3, CD49d, and CD49f antibodies was used to delineate normal eosinophilic maturational patterns. Early stages (eosinophilic promyelocytes/myelocytes) were identified as Siglec-8 dim/CD11b dim to moderate/CD9 dim/CCR3 dim/CD49d bright/CD49f dim, intermediate stages (eosinophilic myelocytes/metamyelocytes) as Siglec-8 moderate/CD11b moderate to bright/CD9 moderate/CCR3 moderate/CD49d moderate/CD49f moderate and mature bands/segmented eosinophils as Siglec-8 bright/CD11b bright/CD9 bright/CCR3 bright/CD49d dim/CD49f bright. Overall maturational patterns were also similar in patients with HES and SM; however, the expression levels of several surface markers were altered compared to normal eosinophils. A novel flow cytometric antibody panel was devised to detect alterations in immunophenotypic patterns of bone marrow eosinophil maturation and evaluated in normal, HES and SM samples. This approach will allow us to elucidate changes in immunophenotypic patterns of bone marrow eosinophilopoiesis in other hematological diseases.

The UDP-glucose/P2Y14 receptor axis promotes eosinophil-dependent large intestinal inflammation.

Ulcerative colitis (UC) is a chronic disorder of the large intestine with inflammation and ulceration. The incidence and prevalence of UC have been rapidly increasing worldwide, but its etiology remains unknown. In patients with UC, the accumulation of eosinophils in the large intestinal mucosa is associated with increased disease activity. However, the molecular mechanism underlying the promotion of intestinal eosinophilia in patients with UC remains poorly understood. Here, we show that uridine diphosphate (UDP)-glucose mediates the eosinophil-dependent promotion of colonic inflammation via the purinergic receptor P2Y14. The expression of P2RY14 mRNA was upregulated in the large intestinal mucosa of patients with UC. The P2Y14 receptor ligand UDP-glucose was increased in the large intestinal tissue of mice administered dextran sodium sulfate (DSS). In addition, P2ry14 deficiency and P2Y14 receptor blockade mitigated DSS-induced colitis. Among the large intestinal immune cells and epithelial cells, eosinophils highly expressed P2ry14 mRNA. P2ry14-/- mice transplanted with wild-type bone marrow eosinophils developed more severe DSS-induced colitis compared with P2ry14-/- mice that received P2ry14-deficient eosinophils. UDP-glucose prolonged the lifespan of eosinophils and promoted gene transcription in the cells through P2Y14 receptor-mediated activation of ERK1/2 signaling. Thus, the UDP-glucose/P2Y14 receptor axis aggravates large intestinal inflammation by accelerating the accumulation and activation of eosinophils.

Effects on hematopoietic and immune system of ultra-high dose rate radiotherapy (FLASH-RT).

166 Background: Ultra-high dose rate radiotherapy, so-called FLASH radiotherapy (FLASH-RT) has shown to reduce radiation-induced damage including lung fibrosis, brain damage, skin fibrosis, etc. We investigated the effect of FLASH-RT compared of conventional dose rate radiotherapy (CONV-RT) at 5Gy of TBI on survival of hematopoietic system, peripheral blood cells, and immune-related responses. Methods: Mice were divided into 3 groups: control (0 Gy), FLASH-RT (109Gy/s), and CONV-RT (0.067Gy/s). FLASH-RT was performed with DIRAMS LINAC producing 6 MeV electron beams. Irradiated mice were sacrificed at 1, 2, 4, 7, 14, 21 and 28 post-TBI of 5 Gy and cellularity of peripheral blood and bone marrow, histological evaluation of immune organ such as spleen and thymus, and T and B lymphocytes in mouse spleen by flow cytometry were investigated. Results: In peripheral blood cell counts, RBC, neutrophil, lymphocyte counts were not significantly different between FLASH-RT and CONV-RT groups from day 1 to day 28 post-TBI. However, FLASH-RT initially induced more apoptosis of bone marrow cells, platelets, and eosinophils of peripheral blood compared to CONV-RT. FLASH-RT and CONV-RT groups showed similar histological changes in the thymus and spleen after 5Gy of TBI, but T lymphocytes of CONV-RT group in day 28 were less recovered compared to those of FLASH-RT group. Our results indicated that FLASH-RT affected more radiation-induced damage of hematopoietic system in early stages compared to CONV-RT, but FLASH-RT showed higher recovery of T lymphocytes after irradiation. Conclusions: These results show that FLASH-RT can induce similar damage and recovery pattern on hematopoietic system with CONV-RT in first week, but FLASH-RT might affect less T-lymphopenia in long-term than CONV-RT.

Type I and II interferons induce granulopoiesis and correlate with organ damage during infection-induced shock

Abstract The mechanisms that drive pathology in infection-induced shock are not well understood. The tick-borne pathogen Ixodes ovatuserhlichia (IOE) induces severe inflammation, vascular damage, liver injury, and death. Mice that lack the receptor for type I IFN, or receptors for both type I and II IFNs, improves survival by 50–70 and 90%, respectively. IOE infection induced a significant increase in circulating granulocytes that depended on both type I and II IFNs. We also observed an IFN-dependent increase in eosinophils and basophils in the bone marrow in IOE-infected C57BL/6TA (WT). Although IOE infection induced an increase in bone marrow IL-5 and GM-CSF levels independent of IFN signaling, bone marrow concentrations of IL-3 and IL-13, both known to promote eosinophil differentiation and survival, increased in an IFNAR-dependent manner. Furthermore, we demonstrate an infection- and IFN-dependent increase of CCL3 and CCL5, two chemokines that promote eosinophil activation and migration. While bone marrow and blood eosinophils and basophils were increased, we observed a specific depletion of eosinophils in WT liver at day 7 post-IOE infection, correlating with increased cell death and severe liver pathology. In contrast, basophil frequencies were increased in IOE-infected WT mice. Additionally, Ifnar-deficient and Ifnar-Ifngr-deficient mice exhibited normal eosinophil and basophil frequencies and reduced liver inflammation, similar to uninfected mice. Together, our data reveal an IFN-driven cytokine and chemokine response that correlates with rampant granulopoiesis and mortality and provide evidence that IFN-induced eosinophil activation may contribute to vascular leak and liver damage in infection-induced shock. Supported by grants from NIH R35GM131842

Regulation of eosinophils by the transcription factor Ikaros

Abstract Immune cell development is a tightly controlled process that enables a homeostatic level of innate and adaptive immune cells of the myeloid and lymphoid lineages, respectively. Imbalance in immune cells can lead to diseases due to immune defects or over-activation. such as immunodeficiency, allergies or autoimmunity. Ikaros is a transcription factor known to be critically important for lymphoid development, with recurrent mutations in different human diseases, but less is known about the role of Ikaros in myeloid development. Ikaros is encoded by the Ikzf1gene, and contains four DNA-binding zinc fingers (ZnF) and two ZnFs mediating protein dimerization. The four DNA-binding ZnFs (ZnF1 through ZnF4) are encoded by three exons, with the two central ZnF2 and ZnF3 encoded by exon 5 being required for binding to the core DNA recognition site. The two flanking ZnFs 1 and 4 are encoded by exon 4 and exon 6, and can modulate DNA binding by contacting the flanking DNA. Interestingly, Ikaros is highly alternatively spliced, creating multiple isoforms with different combinations of the DNA-binding ZnFs. We study the role of Ikaros in immune cell development using Ikzf1-mutant mouse models, and analysis of mice with deletions of exon 4 or exon 6 (encoding ZnF1 and ZnF4, respectively) has revealed differential phenotypes in the two mutant mouse models. Here, we describe a new role of Ikaros in regulation of eosinophils. We have found that Ikaros is required for limiting eosinophil production in bone marrow (BM), as both mutants show increased levels of BM eosinophils. Interestingly, the levels of peripheral tissue eosinophils differ between the two mutants, suggesting a second role of Ikaros in regulating migration, tissue recruitment or retention.

Knockdown of CCR3 gene inhibits Proliferation, migration and degranulation of eosinophils in mice by downregulating the PI3K/Akt pathway.

This study aimed to investigate the effects of CCR3 knockdown (CCR3-/-) on the proliferation, migration, and degranulation of the bone marrow eosinophils (EOS) in mice. Bone marrow cells from wild-type mice (WT) were harvested for primary culture and differentiated into mature EOS, which were then randomly divided into the control, 740Y-P, and LY294002 group. The effects of different concentrations of LY294002 (PI3K inhibitor) and 740Y-P (PI3K agonist) on the proliferation viability of EOS, expressions of EPO, Akt, and p-Akt proteins, and migration changes of EOS were detected. CCR3-/- mice were identified. Then, bone marrow cells of WT and CCR3-/- mice were differentiated into mature EOS and grouped into WT EOS, WT EOS+eotaxin (100ng/mL), CCR3-/- EOS, and CCR3-/- EOS+eotaxin (100ng/mL) group. The changes in EOS proliferation, migration, as well as expressions of EPO, Akt, and p-Akt proteins were detected. The number of migrated cells (P<0.01) and expression of EPO (p<0.05) in the 740Y-P group were higher than those in the control group, while opposite trends were observed for the LY294002 group. Expression levels of p-Akt and Akt in the LY294002 group were significantly lower than in the control group (all P<0.01). Also, the expression of p-Akt in the 740Y-P group was significantly higher than that in the control group (p<0.05). The proliferative activity of EOS, expression of EPO and p-Akt, and the number of migrated cells in the WT EOS group were higher than those in CCR3-/- EOS group (all P<0.05). After adding eotaxin, the WT EOS group was higher than the other three groups (all P<0.05). Mechanistically, CCR3-/- inhibited EOS's proliferation, migration, and degranulation by downregulating PI3K/Akt pathway. This data suggests that the knockout of the CCR3 gene in bone marrow cells may inhibit the function of EOS by downregulating the PI3K/Akt pathway, thereby affecting AR; thus, the CCR3 gene may be a target gene for AR therapy.

Eosinophilia caused by Toxocara canis infection

Eosinophilia is defined as an absolute peripheral blood eosinophil count &gt; 0.5 GL. Most often, its intensity is mild. Eosinophilia usually accompanies other diseases such as allergic, rheumatological, infectious, or oncological. The signs depend on the underlying diseases. After starting treatment of the underlying disease, the eosinophil count returns to the reference range. The diagnosis of eosinophilia starts by excluding the secondary causes. Clonal eosinophilia is a rare disease in which eosinophils are part of a tumor clone. The clonality is confirmed by molecular biology methods. The most common eosinophilic infiltration includes skin, lungs, and heart. The symptoms are not connected with a count of eosinophils in blood or bone marrow. The manifestation can be mild but also severe, life-threatening like venous thromboembolism. This article presents a case of 36-year-old man with eosinophilia caused by Toxocara canis infection manifested by systemic symptoms, erythroderma, and liver failure.

Rapamycin Dampens Inflammatory Properties of Bone Marrow ILC2s in IL-33-Induced Eosinophilic Airway Inflammation.

The alarmin cytokine interleukin (IL)-33 plays an important proinflammatory role in type 2 immunity and can act on type 2 innate lymphoid cells (ILC2s) and type 2 T helper (TH2) cells in eosinophilic inflammation and asthma. The mechanistic target of rapamycin (mTOR) signaling pathway drives immune responses in several inflammatory diseases, but its role in regulating bone marrow responses to IL-33 is unclear. The aim of this study was to determine the role of the mTORC1 signaling pathway in IL-33-induced bone marrow ILC2 responses and its impact on IL-33-induced eosinophilia. Wild-type mice were intranasally exposed to IL-33 only or in combination with the mTORC1 inhibitor, rapamycin, intraperitoneally. Four groups were included in the study: saline-treated (PBS)+PBS, rapamycin+PBS, PBS+IL-33 and rapamycin+IL-33. Bronchoalveolar lavage fluid (BALF), serum and bone marrow cells were collected and analyzed by differential cell count, enzyme-linked immunosorbent assay and flow cytometry. IL-33 induced phosphorylation of the mTORC1 protein rpS6 in bone marrow ILC2s both ex vivo and in vivo. The observed mTOR signal was reduced by rapamycin treatment, indicating the sensitivity of bone marrow ILC2s to mTORC1 inhibition. IL-5 production by ILC2s was reduced in cultures treated with rapamycin before stimulation with IL-33 compared to IL-33 only. Bone marrow and airway eosinophils were reduced in mice given rapamycin before IL-33-exposure compared to mice given IL-33 only. Bone marrow ILC2s responded to IL-33 in vivo with increased mTORC1 activity and rapamycin treatment successfully decreased IL-33-induced eosinophilic inflammation, possibly by inhibition of IL-5-producing bone marrow ILC2s. These findings highlight the importance of investigating specific cells and proinflammatory pathways as potential drivers of inflammatory diseases, including asthma.

Helminth Infections in Children.

Helminth Infections in Children.

Clinical Characteristics and Prognosis of Acute Myeloid Leukemia Patients with inv(16)/t(16;16)(p13.1;q22) and/or CBFβ-MYH11

To summarize the clinical and laboratory characteristics of patients with acute myeloid leukemia (AML) with inv(16)/t(16;16) (p13.1;q22), and to analyze the risk factors affecting the prognosis of the patients. AML patients with inv(16)/t(16;16) (p13.1;q22) and/or CBFβ-MYH11+ admitted to the Department of Hematology, The First Affiliated Hospital of Soochow University from January 1, 2008 to October 30, 2019 were retrospective analyzed, the clinical and laboratory indicators, as well as treatment plans and efficacy evaluations of the patients were all recorded. Furthermore, related factors affecting the overall survival (OS) and event-free survival (EFS) of the patients were analyzed. Among 151 AML patients with inv(16)/t(16;16) (p13.1;q22) and/or CBFβ-MYH11+, the percentage of additional chromosomal abnormalities was about 27.8%, and the most common additional chromosomal abnormality was +22 (33/151, 21.8%), followed by +8 (11/151, 7.3%). There were 112 patients with perfect NGS examination, and the result showed the most common accompanying gene mutations were KIT mutation (34/112, 30.4%) and FLT3 mutation (23/112, 20.5%). Univariate analysis showed that factors affecting EFS included: NE≤0.5×109/L (P=0.006) and combined K-RAS mutation (P=0.002); Factors affecting OS included: Age≥50 years old (P<0.001) and NE≤0.5×109/L (P=0.016). Multivariate analysis showed that NE≤0.5×109/L (P=0.019) was the risk factors affecting OS. The proportion of bone marrow eosinophilia (BME)≥10.00% (P=0.029) was the risk factors affecting EFS. The prognosis for those newly diagnosed AML patients who were of advanced age, the high proportion of bone marrow eosinophils, K-RAS mutations, and agranulocytosis is poor. The treatment plans can be adjusted in the early stage to improve the prognosis of such patients.

Anti-IL5 mepolizumab minimally influences residual blood eosinophils in severe asthma.

Neutralising antibodies against the cytokine interleukin (IL)5 have become widely used for the control of severe eosinophilic asthma. Remarkably, patients receiving neutralising anti-IL5 biological therapies retain a very stable population of residual blood eosinophils. Whether these residual eosinophils are endowed with particular biological activity has not yet been studied, but is of importance in predicting potential long-term effects of IL5 neutralisation in patients. To tackle the effect of IL5 depletion on residual eosinophils, we used a comparative RNA-sequencing approach and compared the gene expression programme of eosinophils arising in IL5-depleted or IL5-replete human or murine hosts, at steady-state in vivo and following in vitro stimulation with the eosinophil-activating alarmin IL33. We compared blood eosinophils from patients with severe allergic eosinophilic asthma treated with anti-IL5 mepolizumab therapy to those of healthy controls and matched asthma patients receiving anti-IgE omalizumab therapy. We made similar comparisons on bone marrow eosinophils from mice genetically deficient or not for IL5. We report that restriction of IL5 availability did not elicit any detectable transcriptional response in steady-state residual eosinophils in mepolizumab-treated patients or IL5-deficient mice, and influenced only a handful of genes in their response to IL33. Together, these results support the notion that treatment with IL5 neutralising antibodies spares a pool of circulating residual eosinophils largely resembling those of healthy individuals.

Eosinophils Upregulate PD-L1 and PD-L2 Expression to Enhance the Immunosuppressive Microenvironment in Multiple Myeloma

Multiple myeloma is a clonal proliferation of malignant plasma cells and in the vast majority of patients remains incurable. The biology of the malignant plasma cells (PCs) in multiple myeloma (MM) is highly influenced by the bone marrow (BM) microenvironment in which they reside. Specifically, BM stromal cells (SCs) have been shown to interact with MM cells to promote their growth, survival and resistance to conventional chemotherapy. In contrast, it remains unclear how the BM microenvironment modulates the response of MM cells to novel agents, such as proteasome inhibitors or responses to innate immune cells. Moreover, it remains unclear which specific cell types within the BM modulate the therapeutic response of MM cells.MM cell lines and patient tumor cells co-cultured with BM and BM-derived eosinophils demonstrated increased growth, proliferation and survival in the presence of the proteasome inhibitors bortezomib and carfilzomib. BM eosinophils have been reported to support the survival of plasma cells by secreting APRIL (a pr oliferation- i nducing l igand), among other growth factors, and contribute to the long-term maintenance of malignant plasma cells. Results indicated that addition of APRIL to MM cultures increased cell proliferation.Targeting immune checkpoints such as programmed cell death protein 1 (PD-1), programmed cell death 1 ligand 1 (PD-L1) and PD-L2 has achieved noteworthy benefit in multiple cancers by blocking immuno-inhibitory signals and enabling patients to produce an effective antitumor response. Programmed death-ligand 1 (PD-L1) and PD-L2 are ligands for the receptor PD-1 within a negative costimulatory network that leads to a T cell exhaustion phenotype, preventing appropriate cellular response to antigens. Treatment with patient BM, BM-derived eosinophils or APRIL led to a substantial upregulation of PD-L1 and PD-L2 on the surface of MM cells (Fig. 1A, B). Furthermore, pre-treatment of MM cells with eosinophils or APRIL suppressed anti-tumor immunity by cytotoxic T lymphocytes (CTLs). shRNA-mediated knockdown of PD-L1 and PD-L2 on MM cells enhanced the cytotoxic effect of CTLs (Fig. 1C). Treatment with the FDA-approved proteasome inhibitors bortezomib, carfilzomib and ixazomib also dramatically and rapidly upregulated PD-L1 and PD-l2 levels on MM cells. Treatment of MM cells with proteasome inhibitors also compromised the anti-myeloma effect of CD8+ T cells. Chemical compound screening was then performed and revealed that small molecule kinase inhibitors that suppressed the basal and drug-induced expression of PD-L1 and PD-L2 on MM cells. Novel small molecule kinase inhibitors that suppressed PD-L1 and PD-L2 on MM cells also triggered CTL activation with antimyeloma benefit.Conclusions: BM-derived eosinophils enhanced immuno-suppressive microenvironmental effects and promoted resistance of MM cells to all proteasome inhibitors tested. APRIL, secreted by eosinophils, promoted MM growth and upregulated the checkpoint proteins PD-L1 and PD-L2 on MM cells. Thus, standard-of-care antimyeloma agents such as proteasome inhibitors as well as the BM-secreted cytokine APRIL suppressed antimyeloma immunity. Antibody-mediated blockade of the PD-L1/PD-L2~PD-1 axis restores antitumor immunity in cancer patients with therapeutic benefit, but exhibits numerous adverse side effects. The development of novel small molecules that suppress checkpoint proteins on MM cells may overcome the drawbacks associated with monoclonal-based checkpoint inhibitors to provide greater specificity, increased tumor response rates and fine-tune therapy while managing adverse events. Small molecules also provide increased oral bioavailability and higher stability at ambient temperature facilitating purification during clinical production. Kinase inhibitors that suppress PD-L1 and PD-L2 to promote antimyeloma immune effects may also limit tumor progression in immunocompetent murine models of myeloma. [Display omitted] DisclosuresMalek:Takeda: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi: Membership on an entity's Board of Directors or advisory committees; Celgene: Speakers Bureau.

Myeloid/lymphoid neoplasms with FLT3 rearrangement.

Myeloid/lymphoid neoplasms (M/LN) with 13q12/FLT3 rearrangement have been suggested as candidates for possible inclusion in the World Health Organization classification group of M/LN with eosinophilia (M/LN-eo). We report 12 patients with confirmed FLT3 rearrangement, six with t(12;13)/ETV6-FLT3; one with ins(13;22)/BCR-FLT3; and five with an unconfirmed partner gene located on chromosome bands 2p16, 3q27, 5q15, 5q35, and 7q36. Disease presentations were heterogeneous, including lymphoblastic leukemia/lymphoma, myeloid sarcoma, chronic eosinophilic leukemia, chronic myelomonocytic leukemia, and myelodysplastic syndrome. However, some common features were observed, such as extramedullary involvement (n = 7, 58%), associated eosinophilia in blood, bone marrow, or tissue (n = 8, 67%), multilineage involvement, either as biphasic myeloid/lymphoid neoplasms (n = 2) or mixed phenotype acute leukemia (n = 2). Mutations were detected in 4/8 (50%) patients by next-generation sequencing. None (0/10) had FLT3 or KIT mutations. Eleven patients received disease-based chemotherapy or hypomethylating agents, three received FLT3 inhibitors, and five patients proceeded to hematopoietic stem cell transplant. Together with a review of 16 cases published in the literature, it is apparent that M/LNs with FLT3 rearrangement show disease features reminiscent of members in the category of M/LN-eo with PDGFRA, PDGFRB, FGFR1, and PCM1/JAK2 rearrangement, characterized by a specific gene rearrangement, frequent eosinophilia, multi-lineage involvement and therapeutic benefit from kinase inhibitors.

More than neutrophils: Lin(+)Ly6G(+)IL-5Rα(+) multipotent myeloid cells (MMCs) are dominant in normal murine bone marrow and retain capacity to differentiate into eosinophils and monocytes.

Bone marrow is a hematopoietic site harboring multiple populations of myeloid cells in different stages of differentiation. Murine bone marrow eosinophils are traditionally identified by Siglec-F(+) staining using flow cytometry, whereas neutrophils are characterized by Ly6G(+) expression. However, using flow cytometry to characterize bone marrow hematopoietic cells in wild-type mice, we found substantial gray areas in identification of these cells. Siglec-F(+) mature eosinophil population constituted only a minority of bone marrow Lin(+)CD45(+) pool (5%). A substantial population of Siglec-F(-) cells was double positive for neutrophil marker Ly6G and eosinophil lineage marker, IL-5Rα. This granulocyte population with mixed neutrophil and eosinophil characteristics is typically attributable to neutrophil pool based on neutral granule staining and expression of Ly6G and myeloid peroxidase. It is distinct from Lineage(-) myeloid progenitors or Siglec-F(+)Ly6G(+) maturing eosinophil precursors, and can be accurately identified by Lineage(+) staining and positive expression of markers IL-5Rα and Ly6G. At 15-50% of all CD45(+) hematopoietic cells in adult mice (percentage varies by sex and age), this is a surprisingly dominant population, which increases with age in both male and female mice. RNA-seq characterization of these cells revealed a complex immune profile and the capacity to secrete constituents of the extracellular matrix. When sorted from bone marrow, these resident cells had neutrophilic phenotype but readily acquired all characteristics of eosinophils when cultured with G-CSF or IL-5, including expression of Siglec-F and granular proteins (Epx, Mbp). Surprisingly, these cells were also able to differentiate into Ly6C(+) monocytes when cultured with M-CSF. Herein described is the discovery of an unexpected hematopoietic flexibility of a dominant population of multipotent myeloid cells, typically categorized as neutrophils, but with the previously unknown plasticity to contribute to mature pools of eosinophils and monocytes.

Patients with mast cell activation symptoms and elevated baseline serum tryptase level have unique bone marrow morphology

Patients with mast cell (MC) activation symptoms and elevated baseline serum tryptase level (MCAS-T) may not necessarily have a clonal MC disorder. Many are diagnosed with hereditary α-tryptasemia (HαT), a genetic trait characterized by autosomal dominant inheritance of multiple copies of TPSAB1 encoding α-tryptase and increased risk for severe anaphylaxis. The aim of our study was to identify and characterize bone marrow MC histopathologic features specific for MCAS-T. A total of 43 patients with MCAS-T underwent evaluation, including bone marrow biopsy, for a MC disorder. The results of the work-up for clonal MC disorders such as systemic mastocytosis and monoclonal MC activation syndrome were negative. Bone marrow MC histopathology was reviewed to identify characteristic features of MCAS-T. Asubgroup of patients was available for tryptase genotyping. Patients with MCAS-T showed unique morphologic and histologic features when compared with controls. MCs were larger (P< .01), hypogranular (P< .01), frequently detected in paratrabecular (P< .05) and perivascular (P< .01) locations, and associated with bone marrow eosinophilia (P< .01). Atotal of 10 patients who were available for tryptase genotyping were all confirmed to have HαT. This subgroup was representative of the larger MCAS-T cohort. We report unique bone marrow MC phenotypic and histopathologic changes in patients with MCAS-T. These morphologic changes are associated with an elevated tryptase level that has been confirmed to be caused by HαT in all patients available for testing.

Airways exposure of bacterial superantigen SEB enhances bone marrow eosinophil population and facilitates its egress to blood and lung tissue

BackgroundDifferentiation of bone marrow eosinophils (BM-EO) and its trafficking to peripheral blood and respiratory mucosa are a hallmark of inflammatory diseases. Staphylococcal enterotoxin B (SEB) has been shown to aggravate airways eosinophilic inflammation. This study aimed to investigate the effects of mouse airways SEB exposure on BM-EO population, as well as its adhesive properties and release of cytokines/chemokines that orchestrate BM-EO trafficking to lungs. MethodsMale BALB/c mice were intranasally exposed to SEB (1 μg), and at 4, 16, 24 and 48 h thereafter, bone marrow (BM), circulating blood and bronchoalveolar lavage (BAL) fluid were collected. Levels of cytokines/chemokines and expressions of VLA-4 and CCR3 in BM were evaluated. Adhesion of BM to ICAM-1 and VCAM-1 were also evaluated. ResultsSEB exposure promoted a marked eosinophil influx to BAL at 16 and 24 h after exposure, which was accompanied by significant increases in counts of immature (16 h) and mature (4 to 48 h) forms of eosinophil in BM, along with blood eosinophilia (16 h). In BM, higher levels of eotaxin, IL-5, IL-4, IL-3 and IL-7 were detected at 16 to 48 h. SEB also significantly increased CCR3 expression and calcium levels in BM-EO, and enhanced the cell adhesion to ICAM-1 (24 h) and ICAM-1 (48 h). ConclusionAirways SEB exposure increases the number of eosinophils in BM by mechanisms involving a network of cytokine and chemokine release, facilitating the BM-EO adhesion to ICAM-1 and VCAM-1 to gain access to the peripheral blood and lung tissues.