Lymphoid Phenotype Research Articles

CXCL-13 serum levels in patients with rheumatoid arthritis treated with abatacept

C-X-C motif chemokine 13 (CXCL-13), which is expressed by synovial follicular dendritic cells and activated mature antigen-experienced T-helper cells, has been described as a surrogate marker of lymphoid phenotype of synovitis in rheumatoid arthritis (RA). A preferential response to anti-interleukin-6 receptor (IL-6R) as compared to anti-tumour necrosis factor alpha (TNFα) monotherapy has been described in patients with increased levels of CXCL-13. We hypothesised that serum levels of CXCL-13 could be used as a biomarker of response to treatment with abatacept (ABA), a T-cell co-stimulation blocker. Serum levels of CXCL-13 and of soluble intracellular adhesion molecule 1 (sICAM-1) (a putative marker of the myeloid subtype of synovitis) were measured by indirect solid-phase enzyme immunoassays, before (T0) and after 6 months of therapy with ABA (T6) in 63 patients with RA. Circulating T follicular helper cells and B cell subpopulations were identified by flow-cytometry. At T0, CXCL-13 serum levels were higher in RA patients than in healthy controls (p=0.0001) and correlated with disease activity, while no difference between the two groups was observed as far as sICAM-1 levels. Serum levels of CXCL-13 levels decreased after therapy with ABA both in patients who achieved a clinical response (p<0.01) and in non-responders (p=0.01), whereas sICAM-1 levels did not significantly change. When comparing RA patients who responded to ABA with non-responders no significant difference of baseline serum levels of CXCL-13 was observed. CXCL-13 serum levels are raised in RA patients and decrease after therapy with ABA. We were not able to demonstrate that serum CXCL-13 levels predict the clinical response to ABA in RA patients.

Advanced Phases at Diagnosis of Childhood Chronic Myeloid Leukemia : The Experience of the International Registry for Chronic Myeloid Leukemia (CML) in Children and Adolescents (I-CML-Ped Study)

Introduction. Chronic myeloid leukemia (CML) is a very rare disease in children and adolescents. The international registry of CML in children and adolescents (I-CML-Ped Study) established in the year 2011 has proven very beneficial to better describe this disease in pediatric cohorts.Aims: To describe the characteristics and the outcome of the pediatric population with advanced phases of CML at initial diagnosis.Patients and Methods: Patients enrolled in the international registry for CML in children and adolescents (I-CML-Ped Study) were the subjects of the study .Disease phases were defined according to the ELN criteria. Characteristics and outcome were determined in children in accelerated (AP) and blastic phase (BP). No recommendations were given regarding the treatment of the patients. Overall survival (OS) was estimated from diagnosis by the Kaplan and Meier method.Results. 479 children and adolescents less than 18 years old with CML were enrolled in the I-CML-Ped study between January 2011 and April 2017. Among them, 37 patients (7.7%) presented at initial diagnosis with CML in advanced phase according to the ELN criteria: 20 children (13 boys and 7 girls, median age 12.6 years - range 3 to 18 years) and 17 children (11 boys and 6 girls, median age 11 years - range 5 to 17 years) were diagnosed in AP and in BP, respectively. Among the 17 patients diagnosed in BP, 12 (70%) had lymphoid and 2 myeloid phenotype (not specified: 2 patients with extramedullary disease as unique criteria of BP). According to the Sokal score for patients less than 45 years, 73% and 82% of the patients in AP and BP at diagnosis were high risk patients, respectively. For the 17 children in BP, treatment consisted of tyrosine kinase inhibitors (TKI) (n=3) or a combination of chemotherapy and TKI successively and/or simultaneously (n=14); 11 of them were transplanted (sibling donor: n=2; unrelated donor n=8; unknown: n=1) with a median time after diagnosis of 7 months (range, 4.5 to 10 months). For the 20 children in AP, treatment consisted of TKI (n=17) or a combination of chemotherapy and TKI successively and/or simultaneously (n=3); 6 of them were transplanted (sibling donor: n=5; unrelated donor n=1) with a median time after diagnosis of 8 months (range, 4 to 21.5 months). With a median time from diagnosis of 3.5 years (range 8 months to 13.5 years), 13 out of 17 patients in BP at initial diagnosis are alive in at least major molecular response (MMR) (11 patients) or without MMR (2 patients) including 5 patients who were not transplanted; death occurred in 3 transplanted patients (relapses: 2 patients; chronic graft versus host disease: 1 patient) and in one patient with progression of the disease. Nineteen out of 20 patients in AP at initial diagnosis are alive (one patient died after progression) in at least MMR (15 patients) or without MMR (2 patients) or unknown status (2 patients). The 5 years OS rates were 94% (95% confidence interval (CI): 66%-99%) and 74% (95% CI: 44%-89%) for patients diagnosed in AP or BP, respectively.Conclusion: The data indicates that the frequency of advanced phases at diagnosis of CML is similar in children and in adults but with a predominance of lymphoid phenotype in children in blastic phase compared to adult population. Hematopoietic stem cell transplantation remains a curative treatment of children in blastic phase at diagnosis.Acknowledgment: The I-CML Ped study is supported by an unrestricted grant from Novartis Pharmaceutical Company DisclosuresNo relevant conflicts of interest to declare.

Engineering of Induced Pluripotent Stem Cell-Derived Hemogenic Endothelium

Background: Autologous cellular therapy of hematological malignancies and primary immunodeficiencies has been limited by the inability to produce functional hematopoietic stem cells (HSCs) from patient-derived induced pluripotent stem cells (iPS). Previous approaches have failed to demonstrate multi-lineage potential or secondary bone marrow engraftment in murine hosts - the gold standards for demonstrating HSC self-renewal and differentiation. Building upon recent evidence that HSCs derive from the definitive hemogenic endothelium (HE) of the embryonic lateral plate mesoderm, we set out to engineer iPS-derived HSCs by directed differentiation, and to investigate potential applications for modeling genetic blood diseases in vitro .Methods: Following embryoid body formation, human iPS derived from bone marrow mesenchymal stem cells underwent stepwise, morphogen-directed differentiation into HE, followed by polycistronic lentiviral transduction with five transcription factors (RUNX1, ERG, LCOR, HOXA5, HOXA9) on day 3 of endothelial-to-hematopoietic transition (EHT) induction, to enable further conversion to HSC-like cells with lymphoid potential in vitro and known multi-lineage engraftment capacity in vivo . A candidate screen of 17 cytokines, epigenetic modifiers and small molecules aimed at inducing HE proliferation was conducted during EHT induction in a RUNX1c gene reporter assay. To model human disease, dermal fibroblast-derived iPS from a hypomorphic RAG2 Omenn syndrome patient were differentiated into HSCs, and cocultured with MS5 or OP9-DL1 stromal cells to induce B or T cell development. Fluorescence-activated cell sorting (FACS) was performed weekly for 6 weeks after initiating lymphoid differentiation.Results: In the candidate screen, IFN-γ enhanced RUNX1c reporter 2.4-fold during EHT, which was confirmed by flow cytometry showing increased hematopoietic progenitor cells (CD34+CD45+). Several compounds were suppressive to EHT, including IFN-α, which abrogated EHT enhancement when combined with IFN-γ. Despite 1 week of lymphoid-directed differentiation, cell populations derived from mutant RAG2 iPS cells showed a predominance of myeloid progenitors (CD38+CD45+CD45RA+), with only 5% of progenitors manifesting a lymphoid phenotype (CD10+CD34+CD45RA+). In contrast, wildtype iPS-derived HSCs produced CD19+IgM- B cell progenitors as well as mature IgM+ B cells (8% versus .06% of CD19+CD21+ B cells), which were comparable to cord blood controls; CD3+CD4+CD8+ T cells were also identified after 3 weeks. Ultimately, we determined that RAG2 -mutant iPS were unable to generate CD19+ B cells and arrested at the early T cell stage (CD5+CD4-CD8-).Conclusion: We determined that transient exposure to IFN-γ greatly improved the EHT of HE, which may bolster the typically low yield of iPSC-derived HSCs. In a lymphoid differentiation assay, wildtype iPS-derived HE were comparable to CB in the formation of mature and immature B/T cells, whereas RAG2 -derived HEs arrested at the pro-thymocyte stage. This finding suggests that our approach to creating engraftable HSCs holds significant potential for modeling hematopoietic disease, both in animal models and as a “disease-on-a-dish,” and for developing therapeutic strategies in genetic blood disorders. DisclosuresNo relevant conflicts of interest to declare.

Major Differences in Lymphocyte Subpopulations Between Cerebrospinal Fluid and Peripheral Blood in Non-Hodgkin Lymphoma Without Leptomeningeal Involvement: Flow Cytometry Evidence of a Cerebral Lymphatic System.

Cerebrospinal fluid (CSF) flow cytometry has a crucial role in the diagnosis of leptomeningeal disease in onco-hematology. This report describes the flow cytometry characterization of 138 CSF samples from patients affected by non-Hodgkin lymphoma, negative for disease infiltration. The aim was to focus on the CSF non-neoplastic population, to compare the cellular composition of the CSF with paired peripheral blood samples and to document the feasibility of flow cytometry in hypocellular samples. Despite the extremely low cell count (1 cell/µl, range 1.0–35) the study was successfully conducted in 95% of the samples. T lymphocytes were the most abundant subset in CSF (77%; range 20–100%) with a predominance of CD4-positive over CD8-positive T cells (CD4/CD8 ratio = 2) together with a minority of monocytes (15%; range 0–70%). No B cells were identified in 90% of samples. Of relevance, a normal, non-clonal B-cell population was documented in 5/7 (71%) patients with primary central nervous system lymphoma at diagnosis (p<0.0001), suggesting a possible involvement of blood-brain barrier cell permeability in the pathogenesis of cerebral B-cell lymphomas. The highly significant differences between CSF and paired peripheral blood lymphoid phenotype (p<0.0001) confirms the existence of an active mechanism of lymphoid migration through the meninges.

A history of microbial exposures has a temporally limited inhibitory effect on mouse lung ILC2 responses to a fungal allergen

Abstract The ability of some microbes and their products to restrain the immune response to allergens has been extensively studied and is a key element of the hygiene hypothesis. However, it is unclear whether a modulated immune response relates to recent microbial exposure, or whether a diverse infectious history promotes sustained changes in reactivity to allergens. Thus, we explored how prior microbial experience in “dirty” mice affected innate immune responses to an airway fungal allergen. Dirty mice were generated by cohousing laboratory mice with pet store mice for at least 60 days to allow for physiological transmission of microbes. Despite changes induced in many immune cell populations, lung type 2 innate lymphoid cell (ILC2) numbers and phenotype were unaltered by cohousing. Short term (4.5 hour) production of the type 2 cytokines IL-5 and IL-13 in response to Alternaria alternata extract was consistently depressed in dirty mice relative to specific pathogen free (SPF) and germ-free animals. This correlated with diminished release of IL-33, an alarmin that activates ILC2, in dirty mice. Nevertheless, at a later timepoint (24 hours) or after repeated Alternaria exposure, the influx of eosinophils, neutrophils and T cells into the lungs was similar in dirty and SPF mice. Our data therefore indicate that a history of robust and diverse microbial exposure delays but ultimately does not restrain development of a type 2 response to airway allergens, in contrast to the established inhibitory effects of acute exposure to certain microbes and their products.

Immune profiling of the bone marrow microenvironment in patients with high-risk localized prostate cancer.

Bone marrow (BM) is a primary metastatic site in prostate cancer (PC) and bone invasion is considered incurable. T cell-mediated immune surveillance is essential in controlling both tumorigenesis and initiation of metastases. Beside tropism, dissemination of PC cells to the BM may be facilitated by defects in BM immune homeostasis predisposing this niche to colonization.To evaluate the BM immune microenvironment in locally advanced, non-metastatic PC, we performed flow cytometry analysis of myeloid and lymphoid subsets in BM aspirates and peripheral blood collected during prostatectomy. Healthy BM aspirates served to establish a reference range for comparison.We found alterations in BM immune composition of PC patients, including an increased CD4/CD8 ratio, enrichment of CD4+ T cells, increased CD56+CD3+ NKT and CD56+CD3- NK yields compared to healthy controls. The lymphoid phenotype remained comparable regarding T cell activation and chemokine receptor-based polarization patterns. Additionally, we found increased B7H3 expression in the myeloid monocyte/macrophage subset and decreased DC infiltration in BM of PC patients.These findings suggest that alterations in the immune milieu may limit immune surveillance that compromise the ability of the BM microenvironment to prevent tumor dissemination, and predispose development of bone metastases in a subset of patients with localized PC.

CML-245: Central Nervous System Restricted Lymphoid Blast Phase Transformation of CML

Objective We describe the clinical manifestations and management of a patient with lymphoid blast transformation of CML with sole compromise of central nervous system. Design case report Setting Hematology ward of Hospital San Juan de Dios, Caja Costarricense Seguro Social, Costa Rica Patients or other participants A 65-year-old male with chronic myelogenous leukemia with a history of non-adherence to TKI. Due to not reaching the European LeukemiaNet milestones, the patient was switched from imatinib to nilotinib. After three months on nilotinib, the patient complained of leg weakness and deafness. Due to persisting neurological symptoms, lumbar puncture was performed. Cerebrospinal fluid flow cytometry demonstrated 92% lymphoblasts. Interventions Full blood count was normal. Bone marrow aspiration showed no evidence of blast phase transformation, polymerase chain reaction showed 17% of BCR ABL transcripts. Head and spine CT and MRI showed no lesions. Tyrosine kinase inhibitor change to dasatinib was solicited. Systemic combination chemotherapy was started. The patient received induction with weekly vincristine and dexamethasone. Anthracycline was omitted due to cardiac disfunction. Triple intrathecal chemotherapy with was administered twice a week until the spinal tap analysis showed no blast presence by flow cytometry. The patient continued on a BFM-like protocol with high dose methotrexate of which he has received four courses. Results In his last follow-up visit the patient had a normal blood count. His last PCR for BCR ABL transcripts had failed to achieve molecular response. BCR ABL mutation study showed no evidence of mutations, medication non-adherence was suspected and addressed. His last spinal tap showed no evidence of blast presence by flow cytometry, his bone marrow aspiration showed no evidence of lymphoblasts. Conclusions The progression to blast phase is a rare event, most of the time related to the development of resistance to tyrosine kinase inhibitors. Less than 20 cases are described in the literature. Central nervous system is the most frequent location. Blast crisis usually presents with a lymphoid phenotype, the frequency of ABL mutations is usually low. The prognosis in these cases is usually dismal with overall survival less than six months.

FRI0105 EVALUATION OF CXCL13 AND ICAM1 SERUM LEVELS AS PREDICTORS OF CLINICAL RESPONSE TO ABATACEPT IN RHEUMATOID ARTHRITIS.

Background:Soluble intercellular adhesion molecule 1 (ICAM1) and C-X-C motif chemokine 13 (CXCL13) were described as differentially associated with two major subtypes of synovitis in rheumatoid arthritis (RA). Raised serum levels of ICAM1 (which is upregulated in synovial fibroblasts in response to TNFα), and of CXCL13 (which is expressed by synovial follicular dendritic cells and activated mature antigen-experienced T-helper cells), are associated with a myeloid or lymphoid synovial phenotype, respectively (1). It has been suggested that a preferential clinical response to anti-TNFα, as compared to anti-IL-6R monotherapy, can be predicted by measuring these two biomarkers (2). No information is available on the possible utility of these biomarkers in RA patients treated with abatacept (ABA), a T-cell co-stimulation blocker.Objectives:To analyze the effect of ABA on ICAM1 and CXCL13 serum levels in RA and to verify whether they predict the response to the drug.Methods:63 RA patients [F/M=51/12; median (10th-90thpercentile) age=60 (41-72) years; CRP-DAS28=4.6 (3.3-5.8); ACPA positive: 86%], before and after 6 months of treatment with ABA + methotrexate and 22 sex and age-matched healthy controls (HC) were evaluated. Serum ICAM1 and CXCL13 levels were dosed by commercial ELISA (Life Technologies and R&amp;D). Response to treatment was defined with the EULAR criteria.Results:CXCL13 serum levels were higher in RA at baseline than in HC [136 (42-325) vs 32 (19-57) pg/ml, p&lt;0.01], while no difference was observed in ICAM1 [186 (125-276) vs 184 (153-246) ng/ml, p=0.9]; positive correlations between ICAM1 and CRP (r:0.28; p=0.03) and CXCL13 levels and CRP (r:0.40; p&lt;0.01) and CRP-DAS28 values (r:0.27, p=0.05) were found. After therapy with ABA, a reduction of CXCL13 was observed [136 (42-325) vs 94 (29-319) pg/ml, p&lt;0.01], both in responders [n: 37: 151 (57-462) vs 97 (26-329) pg/ml; p&lt;0.01] and non-responders (n: 14: 142 (68-293) vs 89 (42-198) pg/ml; p=0.01]. Not significant variation of ICAM1 serum levels was found in the entire cohort [186 (125-276) vs 190 (113-252) ng/ml, p=0.06]. However, a significant decrease was observed in non-responders [222 (169-302) vs 186 (110-233) ng/ml, p=0.02]. At baseline, no significant difference was found among patients seropositive for ACPA if compared with the negative ones [ACPA+ vs ACPA- for ICAM1 [187 (123-280) vs 177 (134-258) ng/ml; p=0.7] and for CXCL13 [143 (42-368) vs 113 (56-270) pg/ml; p=0.4]].Conclusion:Our results confirmed that CXCL13 serum levels are directly correlated with disease activity and demonstrated that ABA therapy induces their reduction. These findings suggest that the co-stimulation blockade at central level and/or in the synovium lead to a reduced production of CXCL13. We could not demonstrate that CXCL13 levels predict the clinical response to ABA in this cohort of patients.

Fibroblast subtypes in tissues affected by autoimmunity: with lessons from lymph node fibroblasts.

The recent advent of single-cell technologies has fast-tracked the discovery of multiple fibroblast subsets in tissues affected by autoimmune disease. In recent years, interest in lymph node fibroblasts that support and regulate immune cells has also grown, leading to an expanding framework of stromal cell subsets with distinct spatial, transcriptional, and functional characteristics. Inflammation can drive tissue fibroblasts to adopt a lymphoid tissue stromal cell phenotype, suggesting that fibroblasts in diseased tissues can have counterparts in lymphoid tissues. Here, we examine fibroblast subsets in tissues affected by autoimmunity in the context of knowledge gained from studies on lymph node fibroblasts, with the ultimate aim to better understand stromal cell heterogeneity in these immunologically reactive tissues.

Conditional Immortalization of Lymphoid Progenitors via Tetracycline-Regulated LMO2 Expression.

Conditional immortalization of hematopoietic progenitors through lentiviral expression of selected transcription factors in hematopoietic stem and progenitor cells provides a promising tool to study stem cell and leukemia biology. In this study, to generate conditionally immortalized lymphoid progenitor (ciLP) cell lines, murine hematopoietic progenitor cells were transduced with an inducible lentiviral "all-in-one" vector expressing LMO2 under doxycycline (DOX) stimulation and the reverse tetracycline-regulated transactivator (rtTA3). For selection of LMO2-expressing ciLPs (LMO2-ciLPs) and longitudinal manipulation in T cell differentiation lymphoid conditions, we developed a robust approach based on coculture with OP9-DL1 stromal cells and improved cytokine conditions allowing a controlled balance between cell proliferation and differentiation in vitro. LMO2-ciLP cell lines with the highest proliferation, vector copy number, and similar insertion pattern were selected for LMO2 "on/off" in vitro study. LMO2 expression under DOX induction resulted in a double negative (DN) 2 differentiation arrest and a propagation of CD44+CD25- myeloid cell population characterized by lymphoid and myeloid phenotypes, respectively. Both DN2 and CD44+CD25- myeloid cell subpopulations expressed c-KIT, suggesting that LMO2-ciLPs were similar to uncommitted progenitors under DOX supplementation. DOX removal resulted in cessation of ectopic LMO2 expression and LMO2-ciLPs continued T cell lymphoid differentiation accompanied by c-KIT downregulation and interleukin 7 receptor expression. Switching off LMO2 expression was accompanied by increased Notch signaling and significant reduction of the CD44+CD25- myeloid cell population under T cell differentiation lymphoid conditions. Although vector insertions in cooperation with LMO2 expression could influence the fate of LMO2-ciLPs and additional experiments are required to evaluate it, our approach provides a promising tool to investigate mechanisms underlying stem cell, leukemia, and lymphocyte biology, leading to novel approaches for disease modeling and therapy evaluation.

Pulmonary Group 2 Innate Lymphoid Cell Phenotype Is Context Specific: Determining the Effect of Strain, Location, and Stimuli.

Group 2 innate lymphoid cells (ILC2s) are enriched at mucosal sites, including the lung, and play a central role in type 2 immunity and maintaining tissue homeostasis. As a result, since their discovery in 2010, research into ILC2s has increased markedly. Numerous strategies have been used to define ILC2s by flow cytometry, often utilizing different combinations of surface markers despite their expression being variable and context-dependent. In this study, we sought to generate a comprehensive characterization of pulmonary ILC2s, identifying stable and context specific markers from different pulmonary compartments following different airway exposures in different strains of mice. Our analysis revealed that pulmonary ILC2 surface marker phenotype is heterogeneous and is influenced by mouse strain, pulmonary location, in vivo treatment/exposure and ex vivo stimulation. Therefore, we propose that a lineage negative cell expressing CD45 and Gata3 defines an ILC2, and subsequent surface marker expression should be used to describe their phenotype in context-specific scenarios.

BRAF V600E and Pten deletion in mice produces a histiocytic disorder with features of Langerhans cell histiocytosis.

Langerhans cell histiocytosis (LCH) is characterized by the accumulation of Langerin (CD207)-expressing histiocytes. Mutational activation of mitogen-activated protein kinase pathway genes, in particular BRAF, drives most cases. To test whether activated BRAF is sufficient for the development of LCH, we engineered mice to express BRAF V600E under the control of the human Langerin promoter. These mice have shortened survivals, smaller lymphoid organs, absent Leydig cells, and fewer epidermal LCs than controls, but do not accumulate histiocytes. To test whether the absence of histiocyte proliferation could be due to oncogene-induced senescence, we engineered homozygous Pten loss in the same cells that expressed BRAF V600E. Like mice with intact Pten, these mice have shortened survivals, smaller thymi, and absent Leydig cells. However, loss of Pten also leads to the accumulation of CD207+ histiocytes in spleen, thymus, and some lymph nodes. While many CD207+ histiocytes in the thymus are CD8-, reminiscent of LCH cells, the CD207+ histiocytes in the spleen and lymph nodes are CD8+. These mice also accumulate large numbers of CD207- cells in the lamina propria (LP) of the small intestine. Both the lymphoid and LP phenotypes are likely due to human Langerin promoter-driven BRAF V600E expression in resident CD8+ dendritic cells in the former and LP dendritic cells in the latter and confirm that Pten loss is required to overcome inhibitory pathways induced by BRAF V600E expression. The complex phenotype of these mice is a consequence of the multiple murine cell types in which the human Langerin promoter is active.

Epigenetic Regulator Genes Direct the Fate of Multipotent Progenitor Cell of Origin in Lineage Switched MLL-AF4 Leukaemia

SummaryThe translocation MLL-AF4 defines a high-risk subtype of acute leukaemia which, uniquely amongst MLL translocations, is almost exclusively associated with a pro-B lymphoid phenotype. However, the ability to switch lineage at relapse allowed interrogation of the cellular origin and lineage determinants of this leukaemia. The origin of MLL-AF4 lymphoid leukaemia was commonly within a multipotent precursor population. In contrast, relapse as MLL-AF4 AML occurs from either a multipotent progenitor or a committed pro-B leukaemic clone. Lineage switching results from the dysregulation of chromatin modifiers, including the nucleosome remodelling and deacetylation complex, NuRD, completely extinguishing the B lymphoid gene regulatory network, instead establishing a myeloid epigenotype. We propose that, in contrast to mixed phenotype acute leukaemias, lineage switch in MLL-AF4 leukemia is driven and maintained by a reorganised epigenome.

Clinical Characteristics of Patients with Ph+ Mixed Phenotype Acute Leukemia

To investigate the clinical biological characteristics and prognosis of the patients with mixed phenotype acute leukemia with t(9;22)(q34;q11.2) and/or BCRABL1 (Ph+ MPAL). The morphological, immunological, cytogenetic, and molecular features of 33 in patients with Ph+ MPAL were retrospectively analyzed in our center from June 2002 to June 2016 according to the scoring proposal of European Group for the Classification of Acute Leukemia(EGIL )1998 and WHO 2008 criteria. All the cases were either treated with acute lymphoblastic leukemia (ALL) induction regimen or combined chemotherapy regimens for both acute lymphoblastic and acute myeloid leukemia,part of which also received tyrosine kinase inhibitor(TKI) and 5 cases underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT) after complete remission. Ph+ MPAL occurred predominantly in male patients (ratio of M/F was 1.75∶1), and a high WBC counts at diagnosis; the WBC count was higher than 30×109/L in 25 patients( 75.8% )， and appeared higher than 100 ×109/L in 13 patients ( 39.4%). Among all the 33 Ph+MPAL patients, 32 (97.0%) had a myeloid / B-lymphoid (M/B) phenotype， and 1 case(3.0%) had a myeloid/ B-lymphoid/ T-lymphoid/ (M/B/T) phenotype. There was no patients displayed myeloid / T-lymphoid (M/T) or B-lymphoid/ T-lymphoid/ (B/T) phenotype. 19 of all cases(57.6%) met the diagnosis criteria of Ph+MPAL based on EGIL 1998 criteria, while the remaining 14 cases can be diagnosed as Ph+ MPAL by WHO 2008 classification,but excluded as Ph+MAPL by EGIL 1998.Karyotype analysis was successfully performed in 31 cases, and out of them 13 (41.9%) had a sole Ph chromosome, 10 (32.3%) had additional chromosome aberration and Ph chromosome was not found in 8 cases (25.8%) .In 31 patients the fusion gene BCR/ABL (P190、P210) was detected,including 17 (54.8%) cases with the p190 BCR/ABL transcript, 8 (25.8%) cases with the p210 BCR/ABL transcript, 4 (12.9%) expressing both transcripts and 2 (6.5%) without any one of these 2 transcripts. 24 out of 33 patients (77.4%) achieved complete remission after induction therapy. The median time achieving CR was 43(26-98)days. The CR rate of patients treated with and without imatinib after the first inducion treatment was 81.3% and 46.7%,respectively (P+0.05). Within the 17 patients treated with imatinib at induction stage，2 of which became BCR/ABLnegative．At consolidation chemotherapy stage, 9 out of 16 patients became BCR/ABL negative, including 3 patients already subjected to HSCT. The median time reached to BCR/ABL negative was 2.87（1.13-9.20）months. Ph+ MPAL is more common in male, and inclined to high WBC counts at diagnosis. Myeloid/B lymphoid phenotype is more common, and the prognosis of patients with Ph+MPAL is poor. Imatinib and allogeneic hematopoietic stem cell transplantation may improve survival of patients with Ph+MPAL.

Age-specific biological and molecular profiling distinguishes paediatric from adult acute myeloid leukaemias

Acute myeloid leukaemia (AML) affects children and adults of all ages. AML remains one of the major causes of death in children with cancer and for children with AML relapse is the most common cause of death. Here, by modelling AML in vivo we demonstrate that AML is discriminated by the age of the cell of origin. Young cells give rise to myeloid, lymphoid or mixed phenotype acute leukaemia, whereas adult cells give rise exclusively to AML, with a shorter latency. Unlike adult, young AML cells do not remodel the bone marrow stroma. Transcriptional analysis distinguishes young AML by the upregulation of immune pathways. Analysis of human paediatric AML samples recapitulates a paediatric immune cell interaction gene signature, highlighting two genes, RGS10 and FAM26F as prognostically significant. This work advances our understanding of paediatric AML biology, and provides murine models that offer the potential for developing paediatric specific therapeutic strategies.

The Complex Role of KDM6A in B-Cell Development and Function

Loss-of-function mutations in KDM6A, an X-linked H3K27 demethylase, occur recurrently in B-cell lymphoid malignancies, including B-cell acute lymphoblastic leukemia and non-Hodgkin lymphoma. Germline inactivating mutations in KDM6A cause a neurodevelopmental disorder called Kabuki syndrome that is associated with recurrent infections and hypogammaglobulinemia.1 The role of KDM6A in normal B-cell development and function, as well as how the somatic loss of KDM6A contributes to B-cell malignancies, has not been completely defined.To address this issue, we generated a conditional knockout mouse of the KDM6A gene (with LoxP sites flanking the 3rd exon) and crossed these mice with Vav1-Cre transgenic mice to selectively inactivate KDM6A in hematopoietic stem/progenitor cells. We characterized normal hematopoiesis from young (6 to 8 week old) and aged (50 to 55 week old) male and female KDM6A conditional KO mice. We found a significant shift from lymphoid to myeloid differentiation in the bone marrow and peripheral blood of these mice. Young, female KDM6A-null mice had mild splenomegaly. Their spleens had an increased number of neutrophils (Gr-1+CD11b+ cells) and erythrocyte progenitors (CD71+Ter119+ cells) and a decreased number of B-cells (B220+ cells). These changes became more pronounced with age and were specific to the female, homozygous KDM6A knockout mice. Furthermore, analysis of B-cell maturation showed that the loss of KDM6A was associated with decreased immature (B220+IgM+ cells) and mature, resting B-cells (B220+IgD+ cells) in the spleen. Similar changes were present in the bone marrow (decreased B220+IgM+ cells and B220+CD19+ cells) and peripheral blood (decreased B220+IgM+, B220+IgD+ and B220+CD19+ cells).Early B-cell development is also altered in KDM6A-null mice. Flow cytometry showed a decrease in multipotent progenitor cells (MPPs) with a decrease in both common lymphoid progenitors (CLPs) and B cell-biased lymphoid progenitors (BLPs) in young, female KDM6A-null mice bone marrow. Next, we performed flow cytometry to catergorize the Hardy fractions of early B-cell development on bone marrow isolated from young, female KDM6A-null mice. B-cell progenitor analysis (Hardy profiles) showed an increase in Fraction A with a concomitant decrease in Fraction B/C and Fraction D, which was likely indicative of an incomplete block in B-cell differentiation after the Fraction A stage. When bulk bone marrow cells isolated from young, female KDM6A-null mice were plated in methylcellulose supplemented with interleukin-7, we observed a significantly decreased colony formation compared with bone marrow cells isolated from wildtype littermates. This pre-B lymphoid progenitor cell plating phenotype was expected given the flow cytometry results of decreased B-cell progenitors outlined above.We examined the effect of the loss of KDM6A expression on germinal center (GC) formation in the spleen following immunization with NP-CGG (4-Hydroxy-3-nitrophenylacetyl-Chicken Gamma Globulin, Ratio 16). Two weeks after NP-CGG immunization, we observed a significant decrease in follicular B-cells (FO) and a significant increase in GC B-cells as compared to wildtype littermates (Figure 1). The result is significant as GC B-cells are thought to be the cell-of-origin of follicular and DLBCL.To determine if inactivation of KDM6A affected antibody production, we measured IgM, IgG, IgE and IgA levels by ELISA from serum isolated from young, female KDM6A-null mice. Results revealed higher levels of IgM and lower levels of IgG in serum from KDM6A-null mice, which is suggestive of a class switch recombination (CSR) defect. Concordant with this result, we observed that the loss of KDM6A impaired CSR to IgG1 in splenic B cells after in vitro stimulation for three days with lipopolysaccharide (LPS), an anti-CD180 antibody and interleukin-4. Moreover, we observed a striking defect in the production of plasma cells from KDM6A-null B-cells after LPS stimulation. Taken together, our data shows that KDM6A plays an important, but complex, role in B-cell development and that loss of KDM6A impedes the B-cell immune response in a specific manner that may contribute to infection and B-cell malignancies.Stagi S, et al. Epigenetic control of the immune system: a lesson from Kabuki syndrome. Immunol Res. 2016; 64(2):345-359. DisclosuresNo relevant conflicts of interest to declare.

Genomic landscape of adult mixed phenotype acute leukemia (MPAL).

7023 Background: MPAL is a rare subgroup of acute leukemia characterized by both myeloid and lymphoid phenotypes. Genetic basis of MPAL is not well understood. Methods: We studied 31 patients (pts) with MPAL (median age 53) that met 2008 WHO criteria. Bone marrow samples were studied by targeted capture sequencing of 295 genes (median 393x), RNA sequencing, and Infinium methylation EPIC array (Illumina). Mutational landscape was compared to 194 AML, 71 B-ALL, and 6 T-ALL cases. Promoter methylation pattern was compared to the data from 194 AML (TCGA), 505 B-ALL and 101 T-ALL cases (Nordlund et al. Genome Biology. 2013). Results: Eighteen (58%) pts had myeloid-T and 13 (42%) had myeloid-B phenotype. Four pts had t(9;22), 1 had 11q23 rarrangement, and 8 had complex karyotype. MPAL had similar number of mutations with AML but had higher number of mutations than B-ALL or T-ALL. Both AML-type and ALL-type mutations were detected in MPAL, supporting the mixed phenotypic features. However, NPM1, CEBPA and GATA2 mutations were specific to AML and were not found in MPAL. Myeloid-T and myeloid-B showed distinct patterns of mutations, in which DNMT3A, IDH2, NOTCH1, IL7R, and FBXW7 mutations were enriched in myeloid-T whereas RUNX1 mutations were enriched in myeloid-B. Myeloid-T and myeloid-B also showed distinct patterns of promoter methylation. Overall, myeloid-T had more hypermethylated CpG loci than myeloid-B. Genes that have essential role in T-cell receptor (TCR) pathway ( CD3D, CD7, CD247, LCK, PRKCQ, CCR9, and TCL1A) were differentially methylated and differentially expressed between myeloid-T and myeloid-B. RNA sequencing revealed several known translocations such as, NSD1-NUP98, and KMT2A-MLLT4, in addition to the novel fusion proteins such as FOXP1-DNAJC15, RUNX1-NAP1L1, and BCL2-TM9SF3. Unbiased hierarchical clustering of MPAL, AML, B-ALL and T-ALL by promoter methylation revealed that myeloid-T had consistent similarity with T-ALL, while myeloid-B showed random similarity with either B-ALL or AML. Conclusions: MPAL is genetically heterogeneous and myeloid-T and myeloid-B shows distinct patterns of mutations, methylation and gene expressions. Therapy for MPAL may need to be tailored based on the genetic profiles.

Dysregulated Lymphoid Cell Populations in Mouse Models of Systemic Lupus Erythematosus.

Biases in the distribution and phenotype of T, B, and antigen-presenting cell populations are strongly connected to mechanisms of disease development in mouse models of systemic lupus erythematosus (SLE). Here, we describe longitudinal changes in lymphoid and antigen-presenting cell subsets in bone marrow, blood and spleen from two lupus-prone strains (MRL/lpr and B6.Sle1.Sle2.Sle3 tri-congenic mice), and how they integrate in our present understanding of the pathogenesis of the disease. In particular, we focus on (autoreactive) T cell activation patterns in lupus-prone mice. Break of T cell tolerance to chromatin constituents (histone peptides) is key to the development of the disease and is related to T cell intrinsic defects, contributed by genetic susceptibility factors and by extrinsic amplificatory mechanisms, in particular over-stimulation by antigen-presenting cells. We also describe shifts in B cell sub-populations, going from skewed immature B cell populations as an indication of disturbed central and peripheral tolerance checkpoints, to enriched long-lived plasma cells, which are key to persistent autoantibody production in the disease. B cell activation mechanisms in SLE are both T cell-dependent (break of tolerance and production of specific autoantibodies) and -independent (polyclonal B cell activation, production of autoantibodies by long-lived plasma cells). By providing a comprehensive evaluation of B and T cell surface markers in two major mouse models of SLE and a description of their changes before and after disease onset, this review illustrates how the study of lymphoid cell phenotype delivers key information regarding pathogenic pathways and supplies tools to assess the beneficial effects of novel therapeutic interventions.

Novel insights into SLC25A46-related pathologies in a genetic mouse model.

The mitochondrial protein SLC25A46 has been recently identified as a novel pathogenic cause in a wide spectrum of neurological diseases, including inherited optic atrophy, Charcot-Marie-Tooth type 2, Leigh syndrome, progressive myoclonic ataxia and lethal congenital pontocerebellar hypoplasia. SLC25A46 is an outer membrane protein, member of the Solute Carrier 25 (SLC25) family of nuclear genes encoding mitochondrial carriers, with a role in mitochondrial dynamics and cristae maintenance. Here we identified a loss-of-function mutation in the Slc25a46 gene that causes lethal neuropathology in mice. Mutant mice manifest the main clinical features identified in patients, including ataxia, optic atrophy and cerebellar hypoplasia, which were completely rescued by expression of the human ortholog. Histopathological analysis revealed previously unseen lesions, most notably disrupted cytoarchitecture in the cerebellum and retina and prominent abnormalities in the neuromuscular junction. A distinct lymphoid phenotype was also evident. Our mutant mice provide a valid model for understanding the mechanistic basis of the complex SLC25A46-mediated pathologies, as well as for screening potential therapeutic interventions.

The frequency of aberrant lymphoid antigens expression in 202 Iraqi patients with de novo acute myeloid leukemia

Background: Immunophenotyping improves both accuracy and reproducibility of acute leukemia classification and is considered, particularly useful for identifying acute myeloid leukemia (AML) with lymphoid marker expression. The incidence of the aberrant phenotypes in AML is still controversial; incidences as high as 88% have been reported. Objectives: To evaluate the occurrence of aberrant lymphoid phenotypes and to correlate their presence with various French-American-British classification (FAB subtypes), 202 cases of newly diagnosed AML were analyzed for lymphoid markers CD1a, CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD19, CD20, and CD79a. Materials And Methods: Whole blood or bone marrow aspirate of 202 patients with de novo AML was collected in ethylenediaminetetraacetic acid tube and analyzed by flow cytometry using a large panel of fluorochrome-labeled monoclonal antibodies. Identification of blast cells was performed using forward scatter versus side scatter (SSC) parameters and CD45 intensity versus SSC dot plots. An antigen was considered positively expressed when at least 20% of the gated cells expressed that antigen. Results: Eighty-five patients (42%) with de novo AML expressed lymphoid-associated antigens. All AML subtypes demonstrated lymphoid-associated antigens except M7. T-cell aberrancy was the most common comprising 32.2% of the total aberrancy. The most frequently lymphoid antigen aberrantly expressed was CD7 (25.7%), followed by CD4 (22.4%) and CD19 (7.9%). Conclusion: A large number of AML cases showed aberrant lymphoid phenotypes. These lymphoid phenotypes might be associated with different leukemia subtypes. T-cell markers are more common than B-cell markers. CD7 was the most common lymphoid marker aberrantly expressed in AML.