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In this study, we aimed at disclosing the main features of paroxysmal nocturnal hemoglobinuria (PNH) clones, their association with presentation syndromes, and their changes during follow-up. A large-scale, cooperative collection (583 clones from 529 patients) of flow cytometric and clinical data was entered into a national repository. Reason for testing guidelines were provided to the 41 participating laboratories, which followed the 2010 technical recommendations for PNH testing by Borowitz. Subsequently, the 30 second-level laboratories adopted the 2012 guidelines for high-resolution PNH testing, both upon order by the local clinicians and as an independent laboratory initiative in selected cases. Type3 and Type2 PNH clones (total and partial absence of glycosyl-phosphatidyl-inositol-anchor, respectively) were simultaneously present in 54 patients. In these patients, Type3 component was sevenfold larger than Type2 (p 0.001). Frequency distribution analysis of solitary Type3 clone size (N = 442) evidenced two discrete patterns: small (20% of peripheral neutrophils) and large ( 70%) clones. The first pattern was significantly associated with bone marrow failure and myelodysplastic syndromes, the second one with hemolysis, hemoglobinuria, and thrombosis. Pediatric patients (N = 34) showed significant preponderance of small clones and bone marrow failure. The majority of PNH clones involved neutrophils, monocytes, and erythrocytes. Nevertheless, we found clones made exclusively by white cells (N = 13) or erythrocytes (N = 3). Rare cases showed clonal white cells restricted only to monocytes (6 cases) or neutrophils (3 cases). Retesting over 1-year follow-up in 151 cases showed a marked clone size increase in 4 cases and a decrease in 13, demonstrating that early breaking-down of PNH clones is not a rare event (8.6% of cases). This collaborative nationwide study demonstrates a clear-cut difference in size between Type2 and Type3 clones, emphasizes the existence of just two classes of PNH presentations based on Type3 clone size, depicts an asymmetric cellular composition of PNH clones, and documents the possible occurrence of changes in clone size during the follow-up.

Aims In hepatocellular carcinoma (HCC), the regulatory protease Dipeptidyl-peptidase IV (DPPIV/CD26), that possesses pro-apoptotic properties, has been found abnormally regulated. The protease inhibitor SerpinB3, exerting anti-apoptotic activity, has also been described to be upregulated, especially in HCCs with poor prognosis. The aim of this study was to investigate the possible relationship between these two molecules in HCC patients and in experimental models. Materials and methods DPPIV/CD26 and SerpinB3 expression was measured in liver specimens of 67 patients with HCC. HepG2 and Huh7 cells, stably transfected to overexpress SerpinB3, and respective control cells were used to assess biological and metabolic modifications of DPPIV/CD26 activity induced by this serpin. Key findings DPPIV/CD26 and SerpinB3 were localized in the same tumoral areas and both molecules were correlated with the grade of tumor differentiation, with the highest values detected in GI tumors. Cell lines over-expressing SerpinB3 displayed upregulation of DPPIV/CD26, likely as a feedback mechanism, due to the DPPIV/CD26 protease activity inhibition by SerpinB3, as confirmed by the similar behavior induced by the inhibitor Sitagliptin. Moreover, they exhibited lower glycogen storage and higher lipid accumulation, typical effects of DPPIV/CD26. Significance A close connection between SerpinB3 and DPPPIV has been identified, but further studies are required to better understand the mechanism by which these proteins communicate and exert metabolic effects in HCC.

// Antonella Teramo 1, 2, * , Gregorio Barila 1, 2, * , Giulia Calabretto 1, 2 , Chiara Ercolin 1, 2 , Thierry Lamy 3 , Aline Moignet 3 , Mikael Roussel 4 , Cedric Pastoret 4 , Matteo Leoncin 1 , Cristina Gattazzo 1, 2 , Anna Cabrelle 2 , Elisa Boscaro 1 , Sara Teolato 1 , Elisa Pagnin 1 , Tamara Berno 1 , Elena De March 1 , Monica Facco 1, 2 , Francesco Piazza 1, 2 , Livio Trentin 1, 2 , Gianpietro Semenzato 1, 2 and Renato Zambello 1, 2 1 Padua University School of Medicine, Department of Medicine, Hematology and Clinical Immunology Branch, Padua, Italy 2 Venetian Institute of Molecular Medicine (VIMM), Padua, Italy 3 Department of Clinical Hematology, University Hospital of Rennes, Rennes, France 4 Biology Department, University Hospital of Rennes, Rennes, France * These authors have contributed equally to this work Correspondence to: Renato Zambello, email: r.zambello@unipd.it Keywords: large granular lymphocyte leukemia, STAT3 mutation, immunophenotype, neutropenia, fas ligand Received: January 20, 2017 Accepted: May 22, 2017 Published: June 27, 2017 ABSTRACT STAT3 mutations have been described in 30-40% of T-large granular lymphocyte (T-LGL) leukemia patients, leading to STAT3 pathway activation. Considering the heterogeneity of the disease and the several immunophenotypes that LGL clone may express, the aim of this work was to evaluate whether STAT3 mutations might be associated with a distinctive LGL immunophenotype and/or might be indicative for specific clinical features. Our series of cases included a pilot cohort of 101 T-LGL leukemia patients (68 CD8+/CD4- and 33 CD4+/CD8±) from Padua Hematology Unit (Italy) and a validation cohort of additional 20 patients from Rennes Hematology Unit (France). Our results indicate that i) CD8+ T-LGL leukemia patients with CD16+/CD56- immunophenotype identify a subset of patients characterized by the presence of STAT3 mutations and neutropenia, ii) CD4+/CD8± T-LGL leukemia are devoid of STAT3 mutations but characterized by STAT5b mutations, and iii) a correlation exists between STAT3 activation and presence of Fas ligand, this molecule resulting highly expressed in CD8+/CD16+/CD56- patients. Experiments with stimulation and inhibition of STAT3 phosphorylation confirmed this relationship. In conclusion, our data show that T-LGL leukemia with specific molecular and phenotypic patterns is associated with discrete clinical features contributing to get insights into molecular bases accounting for the development of Fas ligand-mediated neutropenia.

Injury of vascular endothelium, crucial in vascular disease, is repaired via circulating endothelial progenitor cells (cEPCs). In hypertension, cEPCs number is reduced and function impaired adding further risk for cardiovascular (CV) events. Angiotensin II (Ang II)-induced oxidative stress (OxSt), accelerates cEPCs senescence. Calcitonin gene-related peptide (CGRP), able to prevent and reverse Ang II-induced cEPCs senescence, is reduced in hypertension and stimulated by the antioxidant and anti-inflammatory heme oxygenase (HO)-1. In essential hypertensive patients olmesartan reduced OxSt and markers of CV remodeling and increased HO-1. This study reports in essential hypertensive patients the effect of 6 months treatment with olmesartan on plasma level of CGRP and number and survival of cEPCs.In 20 essential hypertensive patients treated with olmesartan medoxomil (20 mg per day for 6 months), cEPCs (CD34(+)KDR(+), CD133(+)KDR(+) and CD34(+)CD133(+)KDR(+)) (direct 3-color flow cytometry analysis), apoptosis of cEPCs (CD133(+)KDR(+) cells with Annexin V expression), CGRP determination (ELISA) and HO-1 protein level (western blot) were assessed at baseline and after 3 and 6 months of treatments. Olmesartan normalized blood pressure (P 0.001), increased cEPCs from baseline (CD34(+)KDR(+): P 0.003; CD133(+)KDR(+): P 0.0002; CD34(+)CD133(+)KDR(+): P = 0.0008), reduced cEPCs apoptosis (P 0.001) and increased CGRP (P 0.013) and HO-1 (P = 0.039).These results provide a mechanistic rationale for the olmesartan's antioxidant and anti-inflammatory potential translation toward antiatherosclerotic and antiremodeling effects reported on clinical ground.

The JAK/STAT pathway is altered in T-cell large granular lymphocytic leukemia. In all patients, leukemic LGLs display upregulation of phosphorylated STAT3 (P-STAT3) that activates expression of many antiapoptotic genes. To investigate the mechanisms maintaining STAT3 aberrantly phosphorylated using transcriptional protein and functional assays, we analyzed interleukin (IL)-6 and suppressor of cytokine signaling-3 (SOCS3), 2 key factors of the JAK/STAT pathway that induce and inhibit STAT3 activation, respectively. We showed that IL-6 was highly expressed and released by the patients' peripheral blood LGL-depleted population, accounting for a trans-signaling process. By neutralizing IL-6 or its specific receptor with specific antibodies, a significant reduction of P-STAT3 levels and, consequently, LGL survival was demonstrated. In addition, we found that SOCS3 was down-modulated in LGL and unresponsive to IL-6 stimulation. By treating neoplastic LGLs with a demethylating agent, IL-6-mediated SOCS3 expression was restored with consequent P-STAT3 and myeloid cell leukemia-1 down-modulation. Methylation in the SOCS3 promoter was not detectable, suggesting that an epigenetic inhibition mechanism occurs at a different site. Our data indicate that loss of the inhibitor SOCS3 cooperates with IL-6 to maintain JAK/STAT pathway activation, thus contributing to leukemic LGL survival, and suggest a role of demethylating agents in the treatment of this disorder.

Aim/hypothesis: Monocytes/macrophages play important roles in adipose and vascular tissues and can be polarised as inflammatory M1 or anti inflammatory M2. We sought to analyse monocyte polarisation status in type 2 diabetes which is characterised by chronic inflammation. Methods: We enrolled 60 individuals without diabetes and 53 patients with type 2 diabetes. We quantified standard monocyte subsets defined by cluster of differentiation (CD)14 and CD16. In addition based on the phenotype of polarised macrophages in vitro we characterised and quantified more definite M1 (CD68+CCR2+) and M2 (CX3CR1+CD206+/CD163+) monocytes. We also analysed bone marrow (BM) samples and the effects of granulocyte colony stimulating factor (G CSF) stimulation in diabetic and control individuals. Results: We found no alterations in standard monocyte subsets (classical intermediate and non classical) when comparing groups. For validation of M1 and M2 phenotypes we observed that M2 were enriched in non classical monocytes and had lower TNF a content higher LDL scavenging and lower transendothelial migratory capacity than M1. Diabetic patients displayed an imbalanced M1/M2 ratio compared with the control group attributable to a reduction in M2. The M1/M2 ratio was directly correlated with waist circumference and HbA 1c and among diabetic patients M2 reduction and M1/M2 increase were associated with microangiopathy. A decrease in M2 was also found in the BM from diabetic patients with a relative M2 excess compared with the bloodstream. BM stimulation with G CSF mobilised M2 macrophages in diabetic but not in healthy individuals. Conclusions/interpretation: We show that type 2 diabetes markedly reduces anti inflammatory M2 monocytes through a dysregulation in bone marrow function. This defect may have a negative impact on microangiopathy. © 2013 Springer Verlag Berlin Heidelberg.

Background Diffuse lung diseases promote the development of vascular changes and pulmonary hypertension (PH). Endothelial progenitor cells (EPCs) seem to be involved in pulmonary vascular remodeling. We evaluated circulating and intra-pulmonary EPCs in end-stage lung diseases in relation to pulmonary arterial pressure (PAP). Methods The study included 19 patients affected by different end-stage lung diseases, with or without PH. Six lung donors were considered as control group. EPCs were measured in blood samples taken at the time of transplant from pulmonary arteries and veins (by flow cytometry) as well as in lung specimen sections (by confocal microscopy) and expressed as percentage of total number of cells. Results The amount of EPC in lung specimens was significantly different according to type of disease (p = 0.001). Specifically, a higher number of EPCs was detected in idiopathic pulmonary hypertension and idiopathic pulmonary fibrosis with high (> 25 mm Hg) mean PAP ( p = 0.03 for both) compared with chronic obstructive pulmonary disease and control group. There was a direct correlation between intrapulmonary EPCs and PAP. According to receiver operating characteristic curve analysis, the presence of > 3% EPCs had a 91% sensitivity and 93% specificity in identifying high mean PAP. There were no differences in circulating arterial or venous EPCs among groups. Conclusions Intra-pulmonary EPCs are increased in lung diseases with high PAP, suggesting that EPCs may contribute to vascular remodeling in end-stage pulmonary disease.

To evaluate the impact of endothelial progenitor cells (EPCs), a subset of committed circulatory stem cells, on the development of bronchopulmonary dysplasia (BPD) and other short term outcomes in a cohort of extremely premature newborns.Progenitor cells were quantified by flow cytometry at birth in 36 neonates born=28 weeks of gestation and at 36 postmenstrual weeks in 18 of them. Cells expressing the stemness markers CD34, CD133, or both were defined as circulating progenitor cells (CPCs). EPCs were defined as CPCs co-expressing the endothelial marker KDR.Mean (SD) gestational age and birth weight of the infants studied were 26.2(1.5) weeks and 761.6(171.8) grams, respectively. EPC levels at birth did not differ between infants who subsequently developed BPD (n=9) and those who did not (n=24) [CD34(+)KDR(+) EPCs: 81(34-41) vs 80(56-110), p=0.7] and were not correlated with the duration of mechanical ventilation or O2-dependence, nor with the need of surfactant replacement. Infants with a hemodynamically significant patent ductus arteriosus (PDA) (n=22) had significantly lower EPC levels at birth than those with no PDA (n=11) [CD34(+)KDR(+) cells: 47(34-92) vs 142(84.5-221), p=0.008]. Data from the 18 infants studied both at birth and at 36 postmenstrual weeks showed that, while CPCs sharply decline over time, levels of all EPCs phenotypes are preserved after delivery.Levels of EPCs at birth did not affect the risk of developing BPD in our group of extremely premature neonates. However, the association between low EPC counts at birth and PDA may be clinically relevant, and deserves further studies.

Scquizzato E, Zambello R, Teramo A, Baesso I, Varotto S, Albergoni MP, Boscaro E, Cesaro S, Pillon M, Calore E, Gazzola MV, Semenzato G, Messina C, Trentin L. KIR/HLA-I mismatching and risk of relapse in paediatric patients undergoing non-haploidentical allogeneic haematopoietic stem cell transplantation. Pediatr Transplantation 2011: 15:198–204. © 2010 John Wiley & Sons A/S. Abstract: In HSCT setting, KIR-driven alloreactivity might be better predicted if the donor KIR genotype is considered in addition to the recipient HLA genotype. The prediction of NK cell alloreactivity relies on the missing ligand in the recipient, a scenario that can be found in HLA-identical and non-identical allotransplants. The aim of this study was to investigate at genetic level the prognostic impact of recipient HLA-I lacking for donor KIR on allotransplanted patients outcome. We analysed donors KIR genotype and HLA genotype of 60 paediatric patients who received related (n = 15) or unrelated (n = 45) transplantation. When patients were grouped based on the KIR gene type involved in the KIR/HLA-I mismatch, we did not observe any relapse in the group of patients characterized by mismatches involving only inhibitory KIR. On the contrary, all relapses were observed in patients showing at least one activating gene involved in the mismatch (p

The beneficial effects of estrogens on the cardiovascular system are associated with adverse effects on reproductive tissues. On the basis of previous work indicating a major role for estrogen receptor (ER)-alpha in maintaining cardiovascular health, we evaluated the tissue selectivity of the ER alpha-selective agonist propyl pyrazole triol (PPT) compared with 17beta-estradiol (E2) in vivo. Four weeks postovariectomy, equimolar doses of PPT and E2 were administered to rats in subcutaneous implants for 5 d. Both treatments restored rapid vasorelaxation of aortic tissue to estrogenic agents and prevented coronary hyperresponsiveness to angiotensin II in isolated heart preparations. Accordingly, multiple endpoints of myocardial ischemia-reperfusion injury exacerbated by ovariectomy returned to baseline following treatment. These protective effects were linked to increased in vivo levels of endothelial progenitor cells (EPCs). Human EPC function was enhanced in vitro after PPT treatment. In sharp contrast to E2, PPT treatment had no effect on uterine weight and histomorphology except for vessel density, and failed to up-regulate classic estrogen target genes. Dissection of the effects on vascular reactivity and uterine morphology was also observed following increased exposure to PPT at a higher dose for longer time. These data provide the first in vivo evidence for tissue-specific ER alpha activation. By conferring cardiovascular protection dissected from unwanted uterotrophic effects, ER alpha-selective agonists may represent a potential safer alternative to natural hormones.

Objectives Metabolic syndrome (MetS) associates with endothelial dysfunction and a high risk of cardiovascular events and death. Circulating progenitor cells have been shown to contribute to endothelial homeostasis and repair . We aimed to test whether progenitor cell count is an independent event predictor and modifies cardiovascular risk associated with MetS. Methods On the basis of the expression of CD34, CD133 and KDR, 6 phenotypes of progenitor cells were counted using flow cytometry in 214 subjects with and without MetS. We recorded classical risk factors and MetS components, cumulative risk estimates, and high-sensitive C-reactive protein. Subjects were followed-up for a median of 34 months to collect total events, cardiovascular events and all-cause mortality. Results In the Cox proportional hazards regression analyses, we found that, unlike other phenotypes, reduced CD34+ cells predicted cardiovascular and total events and death, independently of all potential confounders. Remarkably, a low CD34+ cell count significantly increased the risk associated with MetS, as shown by synergy indexes. Conclusion The level of circulating CD34+ cells is a novel independent risk biomarker and modulates outcomes in the MetS, suggesting that generic progenitor cells have a role in disease development or progression over the long-term.

The beneficial or detrimental effects of androgens on the cardiovascular system are debated. Endothelial progenitor cells are bone-marrow-derived cells involved in endothelial healing and angiogenesis, which promote cardiovascular health. Oestrogens are potent stimulators of endothelial progenitor cells, and previous findings have indicated that androgens may improve the biology of these cells as well. In the present study, we show that testosterone and its active metabolite dihydrotestosterone exert no effects on the expansion and function of late endothelial progenitors isolated from the peripheral blood of healthy human adult males, whereas they positively modulate early ‘monocytic’ endothelial progenitor cells. In parallel, we show that castration in rats is followed by a decrease in circulating endothelial progenitor cells, but that testosterone and dihydrotestosterone replacement fails to restore endothelial progenitor cells towards normal levels. This is associated with persistently low oestrogen levels after androgen replacement in castrated rats. In a sample of 62 healthy middle-aged men, we show that circulating endothelial progenitor cell levels are more directly associated with oestradiol, rather than with testosterone, concentrations. In conclusion, our results collectively demonstrate that androgens exert no direct effects on endothelial progenitor cell biology in vitro and in vivo.

We have shown previously that the che- mokine receptors CXCR3 and CXCR6 are coex- pressed by Th1 cells infiltrating the lung and the granuloma of patients with sarcoidosis. In this study, we evaluated the role of CCL20/CCR6 in- teraction in the pathogenesis of acute and chronic pulmonary sarcoidosis. By flow cytometry and mo- lecular analyses, we have demonstrated that Th1 cells isolated from the bronchoalveolar lavage (BAL) of patients with sarcoidosis and T cell alve- olitis are equipped with CCR6. Furthermore, CCR6 T cells coexpressed the chemokine recep- tors CXCR3 and CXCR6. Immunohistochemical analysis of lung specimens has shown that CCR6 T cells infiltrate lung interstitium and surround the central core of the granuloma. It is interesting that CCR6 was never detected on the alveolar macro- phage (AM) surface, and it is observed in the cyto- plasm of AMs from patients with sarcoidosis and alveolitis. The CCR6 ligand CCL20 was expressed by macrophages, multinucleated giant cells, and epithelioid cells infiltrating the granuloma. Fur- thermore, detectable levels of CCL20 protein are seen in the BAL fluid components of patients with active sarcoidosis, and sarcoid AMs release the CCR6 ligand in vitro. From a functional point of view, sarcoid Th1 cells were able to respond to CXCL10, CXCL16, and CCL20 in migratory as- says. In vitro kinetic studies demonstrated that CCR6 is induced rapidly by IL-2, IL-18, and IFN-. In conclusion, T cells expressing CCR6, CXCR3, and CXCR6 act coordinately with respec- tive ligands and Th1 inflammatory cytokines in the alveolitic/granuloma phases of the disease. J. Leu- koc. Biol. 82: 946-955; 2007.

Background Endothelial progenitor cells (EPC) promote angiogenesis and vascular repair. Though reduced EPC levels have been shown in rheumatoid arthritis, no study has so far evaluated EPCs in children with juvenile idiopathic arthritis (JIA). We aimed to study circulating EPCs in children with JIA, their relation to disease activity, and effects of anti TNF-α treatment. Methods Circulating EPCs were quantified by flow cytometry based on CD34, CD133 and KDR expression in peripheral blood of 22 patients with oligoarticular JIA and 29 age-matched controls. EPCs were re-assessed in children with methotrexate-resistant oligo-extended JIA before and up to 12 month after initiation of anti-TNF-alpha therapy. Plasma concentrations of inflammatory and EPC-regulating factors were measured using a multiplex array. Confocal immunofluorescence was used to demonstrate EPCs in synovial tissues. Results Children with active JIA showed a significant reduction of relative and absolute counts of circulating progenitor cells and EPCs compared to age-matched healthy controls. CD34+ cell levels were modestly and inversely correlated to disease activity. A strong inverse correlation was found between serum TNF-α and EPC levels. In 8 patients treated with anti TNF-α agents, the number of EPCs rose to values similar to healthy controls. CD34+KDR+ EPCs were found in the synovial tissue of JIA children, but not in control. Conclusions Children with JIA have reduced levels of the vasculoprotective and proangiogenic EPCs. While EPCs may contribute to synovial tissue remodelling, EPC pauperization may indicate an excess cardiovascular risk if projected later in life.

Introduction: Given the critical role of BCR–ABL kinase activity in chronic myeloid leukemia (CML), tyrosine kinase inhibitors (TKIs) are currently considered the cornerstone of CML treatment. Previous studies have suggested that TKIs may influence anti-tumor immunity through off-target modulation of different immune effectors. Natural killer (NK) cells, as well as T cells in the context of adaptive immunity, are a key component of the innate immune system, providing first-line defense against virally infected cells and tumors. The activity of NK cells is modulated by a finely-tuned balance between signals received from inhibitory and activating cell surface receptors. Aims: We sought to evaluate the impact of first and second generation TKIs on modulating different NK cell receptors patterns; secondly we studied the effect of a TKIs driven NK subpopulations selection on treatment response. Finally, we analyzed the T cells Vβ-TCR repertoire to identify any restrictions. Materials and Methods: Peripheral blood samples from 25, 9 and 8 chronic phase CML patients treated with imatinib frontline, nilotinib and dasatinib as first or second line therapy, respectively, were collected. Patients characteristics are described below (see table 1). After separation of mononuclear cells (PBMC), the expression of several NK cell receptors (Killer Immunoglobulin-like Receptors, KIR: p70, p140, p58/p50; Killer Lectin-like Receptors, KLR: CD94, NKG2A, NKG2C/A, NKG2D; Natural Cytotoxicity Receptors, NCR: NKp30, NKp44, NKp46, NKp80; Co-receptors: 2B4; LIR1/ILT2, GPR56) and Vβ TCR-repertoire were analized by flow cytometry analysis. Treatment response was assessed with standardized real quantitative polymerase chain reaction and cytogenetics according to ELN recommendations. Results: The leukocyte count was not statistically different between groups (WBC = 5.5 x 109 / L vs. 6.8 x 109 / L vs. 5.6 x 109 / L, p = 0.09, respectively); also lymphocytes, considered either in percentage or absolute number, were comparable (32% vs 26% vs 35%, p = 0.08), as well as the percentage and absolute number of NK cells (20%; 0.37 x 109 / L vs. 15%; 0.26 x 109 / L vs. 24%; 0.54 x 109 / L (p = 0.17, p = 0.10).The analysis of NK receptors expression showed that patients treated with Imatinib exhibited a preferential selection of NK cells subpopulations harboring activating receptors (NKp30, NKp46, NKp80 and NKG2D), while in Dasatinib treated patients an increased expression of KIR (KIR2DL1) receptors was observed (figure 1). Interestingly, these effects were documented also in the absence of lymphocytosis. 44.4% (4 of 9 patients) of patients treated with nilotinib showed preferential expression of Vβ chains, compared with 87.5% of patients treated with dasatinib; no TCR-repertoire restriction was documented in the sole TKI primary resistant patient. 8 out of 17 patients showed a preferential expression of more than oneVβ chain (figure 2). No specific NK receptors profiles were found to be associated with different degrees of treatment response. Conclusions: These preliminary data suggest the existence of a different NKRs and T cell receptor repertoire modulation, mediated by Tyrosine-Kinase Inhibitors. Since no significant correlation between response and specific NK receptor profiles has been demonstrated, TKIs immunomodulatory effect seems secondary compared to direct inhibition of BCR-ABL kinase. However, it's conceivable that NK and T cells subpopulations selection, induced by TKIs, may become relevant in the immunological control of leukemic disease at the time of drug discontinuation. These observations are currently being investigated on a larger series of patients. Figure 1 NK cell receptors differentially expressed between imatinib, nilotinib and dasatinib treated patients. Figure 1. NK cell receptors differentially expressed between imatinib, nilotinib and dasatinib treated patients. Figure 2 Figure 2. Figure 3 T cell receptor repertoire in nilotinib (A) and dasatinib (B) treated CML patients Figure 3. T cell receptor repertoire in nilotinib (A) and dasatinib (B) treated CML patients Disclosures No relevant conflicts of interest to declare.

Most patients affected by chronic lymphocytic leukemia are diagnosed by flow cytometry. Several immunophenotypic markers have been identified as significant and independent prognostic variables, especially from retrospective cohorts. However, while attractive because their detection is inexpensive and feasible in most laboratories, only few have been validated by independent series. The expression of leukocyte-associated immunoglobulin-like receptor-1 (also known as LAIR1, LAIR-1 or CD305), an inhibitor of B-cell receptor-mediated signaling, has been reported to be lacking in high-risk chronic lymphocytic leukemia. However, its correlation with biological variables and its prognostic significance remain unknown. We investigated 311 consecutive patients, prospectively enrolled since 2007. Methods for studying patients were standardized and included clinical assessment, immunophenotype, fluorescence in situ hybridization, and status of immunoglobulin heavy chain variable region genes. Overall, 22.1% of patients had Binet stage B or C disease, 38.5% had unmutated immunoglobulin genes, 15.1% had high-risk cytogenetic abnormalities, 23.4% were CD38(+), 37.8% CD49d(+), and 59.8% LAIR1(+). Expression of LAIR1 was inversely related to that of CD38 (P=0.0005), but was not associated with CD49d expression (P=0.96). A significantly lower expression of LAIR1 was observed in patients with Binet stage B or C disease (P=0.023), and in the presence of high-risk cytogenetic abnormalities (P=0.048) or unmutated immunoglobulin heavy chain variable region genes (P

Acromegaly increases cardiovascular risk, possibly due to the high prevalence of classical risk factors. However, in vitro studies show a protective role of GH/IGF-1 on the endothelium.The objective of the study was to investigate circulating endothelial progenitor cells (EPCs), a marker of vascular regeneration, in acromegalic patients and how they are affected by acromegaly treatment.This was a cross-sectional case-control and observational study.The study was conducted at a tertiary ambulatory referral endocrinology center.Forty-three acromegalic patients (26 active; 17 inactive) and 43 control subjects matched by age, gender, and degree of glucose tolerance participated in the study.Circulating EPCs were quantified by flow cytometry based on the expression of CD34, CD133, and kinase insert domain-containing receptor (KDR). Nine patients with active acromegaly were reevaluated after 24 weeks of treatment with somatostatin analogs (SSAs).Differences in EPC levels between patients and controls were measured.Acromegalic patients showed a significant reduction of the total CD34(+)KDR(+) EPC population compared with controls, which was more evident in patients without diabetes or hypertension. More definite CD34(+)CD133(+)KDR(+) EPCs were reduced in patients with active compared with those with inactive acromegaly and compared with controls. The number of CD34(+)CD133(+)KDR(+) EPCs correlated with IGF-1 levels (r = -0.45; P.001), fasting plasma glucose (r = -0.40; P = .004), and the homeostasis model assessment index of insulin resistance (r = -0.32; P = .026). CD34(+)CD133(+)KDR(+) EPCs increased 2-fold after SSA treatment.Acromegalic patients have a reduced endothelial regenerative capacity, possibly due to activation of the GH/IGF-1, rather than concomitant risk factors. Treatment with SSAs can restore immature EPCs to normal levels.

OBJECTIVE Vasculoprotective endothelial progenitor cells (EPCs) are regulated by stromal-derived factor-1α (SDF-1α) and are reduced in type 2 diabetes. Because SDF-1α is a substrate of dipeptidyl-peptidase-4 (DPP-4), we investigated whether the DPP-4 inhibitor sitagliptin modulates EPC levels in type 2 diabetic patients. RESEARCH DESIGN AND METHODS This was a controlled, nonrandomized clinical trial comparing 4-week sitagliptin (n = 16) versus no additional treatment (n = 16) in addition to metformin and/or secretagogues in type 2 diabetic patients. We determined circulating EPC levels and plasma concentrations of SDF-1α, monocyte chemoattractant protein-1 (MCP-1), vascular endothelial growth factor (VEGF), and nitrites/nitrates. RESULTS There was no difference in clinical baseline data between the sitagliptin and control arms. After 4 weeks, as compared with control subjects, patients receiving sitagliptin showed a significant increase in EPCs and SDF-1α and a decrease in MCP-1. CONCLUSIONS Sitagliptin increases circulating EPCs in type 2 diabetic patients with concomitant upregulation of SDF-1α. This ancillary effect of DPP-4 inhibition might have potential favorable cardiovascular implications.

Background Natural killer (NK) cells can bridge innate and acquired immunity, and play a role in autoimmunity. A few studies evaluated the distribution of NK cells and the expression of their receptors in chronic immune-mediated demyelinating polyneuropathies. We investigated NK cell distribution and NK cell receptor expression in 20 naive patients with anti-MAG polyneuropathy (MAG-PN). Methods Using flow cytometry, we analysed NK cells and a series of NK cell receptors in the peripheral blood of patients with MAG-PN, and, as controls, in patients with chronic inflammatory demyelinating peripheral polyradiculoneuropathy (CIDP) and in healthy subjects. Six MAG-PN patients were also tested after rituximab treatment. Results At baseline the percentage of NK cells did not differ among the groups. KIR2DL2 receptor expression in MAG-PN patients was higher, andCD94/NKG2A receptor expression in both MAG-PN and CIDP patients was lower than in healthy controls. These abnormalities did not correlate with any clinical or demographic variable. No modification was found after rituximab therapy. Conclusions The data suggest that MAG-PN shows abnormalities in NK cell receptors that characterise other autoimmune diseases, and cannot help in differential diagnosis with CIDP. The impairment of the relevant CD94/NKG2A inhibitory pathway, which might play a central role in the development and perpetuation of MAG-PN, warrants further functional investigations.

Several cell types contribute to atherosclerotic calcification. Myeloid calcifying cells (MCCs) are monocytes expressing osteocalcin (OC) and bone alkaline phosphatase (BAP). Herein, we tested whether MCCs promote atherosclerotic calcification in vivo. We show that the murine spleen contains OC(+)BAP(+) cells with a phenotype similar to human MCCs, a high expression of adhesion molecules and CD11b, and capacity to calcify in vitro and in vivo. Injection of GFP(+) OC(+)BAP(+) cells into 8- or 40-week ApoE(-/-) mice led to more extensive calcifications in atherosclerotic areas after 24 or 4 weeks, respectively, compared to control OC(-)BAP(-) cells. Despite that OC(+)BAP(+) cells had a selective transendothelial migration capacity, tracking of the GFP signal revealed that presence of injected cells within atherosclerotic areas was an extremely rare event and so GFP mRNA was undetectable by qPCR of lesion extracts. By converse, injected OC(+)BAP(+) cells persisted in the bloodstream and bone marrow up to 24 weeks, suggesting a paracrine effect. Indeed, OC(+)BAP(+) cell-conditioned medium (CM) promoted calcification by cultured vascular smooth muscle cells (VSMC) more than CM from OC(-)BAP(-) cells. A genomic and proteomic investigation of MCCs identified allograft inflammatory factor (AIF)-1 as a potential candidate of this paracrine activity. AIF-1 stimulated VSMC calcification in vitro and monocyte-specific (CD11b-driven) AIF-1 overexpression in ApoE(-/-) mice increased calcium content in atherosclerotic areas. In conclusion, we show that murine OC(+)BAP(+) cells correspond to human MCCs and promote atherosclerotic calcification in ApoE(-/-) mice, through paracrine activity and modulation of resident cells by AIF-1 overexpression.

Bone marrow (BM) derived stem and progenitor cells contribute to cardiovascular homeostasis and are affected by cardiovascular risk factors. We devised a clinical data-driven approach to test candidate stem cell mobilizing mechanisms in pre-clinical models. We found that PB and BM CD34+ cell counts were directly correlated, and that most circulating CD34+ cells were viable, non-proliferating and derived from the BM. Thus, we analyzed PB and BM CD34+ cell levels as a two-compartment model in 72 patients with or without cardiovascular disease. Self-organizing maps showed that disturbed compartmentalization of CD34+ cells was associated with aging and cardiovascular risk factors especially diabetes. High activity of DPP-4, a regulator of the mobilizing chemokine SDF-1α, was associated with altered stem cell compartmentalization. For validation of these findings, we assessed the role of DPP-4 in the BM mobilization response of diabetic rats. Diabetes differentially affected DPP-4 activity in PB and BM and impaired stem/progenitor cell mobilization after ischemia or G-CSF administration. DPP-4 activity in the BM was required for the mobilizing effect of G-CSF, while in PB it blunted ischemia-induced mobilization. Indeed, DPP-4 deficiency restored ischemia (but not G-CSF)-induced stem cell mobilization and improved vascular recovery in diabetic animals. In conclusion, the analysis of stem cell compartmentalization in humans led us to discover mechanisms of BM unresponsiveness in diabetes determined by tissue-specific DPP-4 dysregulation.

OBJECTIVE Diabetes mellitus (DM) increases cardiovascular risk, at least in part, through shortage of vascular regenerative cells derived from the bone marrow (BM). In experimental models, DM causes morphological and functional BM alterations, but information on BM function in human DM is missing. Herein, we sought to assay mobilization of stem and proangiogenic cells in subjects with and without DM. RESEARCH DESIGN AND METHODS In a prospective trial (NCT01102699), we tested BM responsiveness to 5 μg/kg human recombinant granulocyte colony–stimulating factor (hrG-CSF) in 24 individuals with DM (10 type 1 and 14 type 2) and 14 individuals without DM. Before and 24 h after hrG-CSF, we quantified circulating stem/progenitor cells and total and differential white blood cell counts. We also evaluated in vivo the proangiogenic capacity of peripheral blood mononuclear cells using the Matrigel plug assay. RESULTS In response to hrG-CSF, levels of CD34+ cells and other progenitor cell phenotypes increased in subjects without DM. Patients with DM had significantly impaired mobilization of CD34+, CD133+, and CD34+CD133+ hematopoietic stem cells and CD133+KDR+ endothelial progenitors, independently of potential confounders. The in vivo angiogenic capacity of peripheral blood mononuclear cells significantly increased after hrG-CSF in control subjects without DM, but not in patients with DM. DM was also associated with the inability to upregulate CD26/DPP-4 on CD34+ cells, which is required for the mobilizing effect of granulocyte colony–stimulating factor. CONCLUSIONS Stem and proangiogenic cell mobilization in response to hrG-CSF is impaired in DM, possibly because of maladaptive CD26/DPP-4 regulation. These alterations may hamper tissue repair and favor the development of cardiovascular complications.

Diabetes mellitus (DM) alters circulating progenitor cells relevant for the pathophysiology of coronary artery disease (CAD). While endothelial progenitor cells (EPCs) are reduced, there is no data on procalcific polarization of circulating progenitors, which may contribute to vascular calcification in these patients. In a cohort of 107 subjects with and without DM and CAD, we analyzed the pro-calcific versus endothelial differentiation status of circulating CD34+ progenitor cells. Endothelial commitment was determined by expression of VEGFR-2 (KDR) and pro-calcific polarization by expression of osteocalcin (OC) and bone alkaline phosphatase (BAP). We found that DM patients had significantly higher expression of OC and BAP on circulating CD34+ cells than control subjects, especially in the presence of CAD. In patients with DM and CAD, the ratio of OC/KDR, BAP/KDR, and OC+BAP/KDR was about 3-fold increased than in other groups. EPCs cultured from DM patients with CAD occasionally formed structures highly suggestive of calcified nodules, and the expression of osteogenic markers by EPCs from control subjects was significantly increased in response to the toll-like receptor agonist LPS. In conclusion, circulating progenitor cells of diabetic patients show a phenotypic drift toward a pro-calcific phenotype that may be driven by inflammatory signals.

Aims: In diabetes, endothelial damage promotes macroangiopathy and endothelial regeneration is impaired, owing to reduced endothelial progenitor cells (EPCs). Given that insulin influences endothelial biology, we compared the effects of add-on basal insulin analogues on endothelial damage and regeneration in type 2 diabetes (T2D). Methods: This was a 6-month randomized crossover trial comparing add-on insulin detemir versus glargine in poorly controlled T2D with macroangiopathy. At baseline, crossover (3 months) and study end (6 months), we measured HbA1c, EPCs, circulating endothelial cells (CECs), VCAM-1, ICAM-1 and E-selectin. Body weight and hypoglycaemic episodes were also recorded. Results: Forty-two patients completed the study, randomly assigned to the glargine–detemir (n = 21) or the detemir–glargine (n = 21) schedule. At crossover, EPC levels did not change compared with baseline, but significantly increased at study end. CECs decreased over time and were significantly reduced at study end. ICAM-1, VCAM-1 and E-selectin were significantly reduced at crossover and further decreased at study end. No differences were seen in these effects between detemir and glargine. HbA1c showed a carryover effect and its reduction was similar with detemir and glargine in the first arm. Incidence of hypoglycaemia and weight gain was lower with detemir than with glargine in both arms. Conclusion: Optimized glycaemic control by add-on basal insulin improved indexes of endothelial damage and regeneration. Compared to glargine, detemir achieved similar endothelial protection with lower weight gain and less hypoglycaemia. These results might have implications for therapy of aging T2D patients with cardiovascular disease.

Rationale: Acquisition of a procalcific phenotype by resident or circulating cells is important for calcification of atherosclerotic plaques, which is common in diabetes. Objective: We aim to identify and characterize circulating calcifying cells, and to delineate a pathophysiological role for these cells in type 2 diabetes. Methods and Results: We demonstrate for the first time that a distinct subpopulation of circulating cells expressing osteocalcin and bone alkaline phosphatase (OC + BAP + ) has procalcific activity in vitro and in vivo. The study of naïve patients with chronic myeloid leukemia indicated that OC + BAP + cells have a myeloid origin. Myeloid calcifying OC + BAP + cells (MCCs) could be differentiated from peripheral blood mononuclear cells, and generation of MCCs was closely associated with expression of the osteogenic transcription factor Runx2. In gender-mismatched bone marrow–transplanted humans, circulating MCCs had a much longer half-life compared with OC − BAP − cells, suggesting they belong to a stable cell repertoire. The percentage of MCCs was higher in peripheral blood and bone marrow of type 2 diabetic patients compared with controls but was lowered toward normal levels by optimization of glycemic control. Furthermore, diabetic carotid endoarterectomy specimens showed higher degree of calcification and amounts of cells expressing OC and BAP in the α-smooth muscle actin–negative areas surrounding calcified nodules, where CD68 + macrophages colocalize. High glucose increased calcification by MCCs in vitro, and hypoxia may regulate MCC generation in vitro and in vivo. Conclusions: These data identify a novel type of blood-derived procalcific cells potentially involved in atherosclerotic calcification of diabetic patients.

EPCs (endothelial progenitor cells) exert vasculoprotective effects and can be used for regenerative therapies. However, several isolation protocols have been described, with inconsistent results. Statins are among the most effective compounds that stimulate EPC numbers in vivo and ex vivo. We aim to describe the effects of rosuvastatin on different subtypes of putative EPCs. EPCs were cultured from mononuclear cells of blood donors and isolated according to three protocols: CFU-EC (colony forming units-endothelial cells), early (or 'monocytic') EPCs and late outgrown EPCs. Rosuvastatin (0.1-100 nM) was added at the beginning of culture (T0) or after the initial adhesion step (T1). Polarization of monocytic EPCs was assessed as expression of proinflammatory M1 markers (CD68 and CCR2) or anti-inflammatory M2 markers (CX3CR1, CD163, CD206). We found that 1 nM rosuvastatin increased the number of CFU-EC and late EPCs by about 3-fold, while lower concentrations had no significant effects. Rosuvastatin (0.1 nM) increased AcLDL+Lectin+ early EPCs by about 60%, while higher concentrations exerted inhibitory effects on early EPCs. Addition of rosuvastatin at T0 was more effective in stimulating CFU-EC and early EPCs, while addition at T1 was more effective in stimulating late EPCs. Rosuvastatin had no effects on proliferation rate of CFU-EC, early EPCs and late EPCs. We also found that 0.1 nM rosuvastatin reduced the M1/M2 ratio in early EPCs, which retain monocytic features. In conclusion, we show that rosuvastatin had significant stimulatory effects on EPCs irrespective of the culture protocol. Rosuvastatin also induced anti-inflammatory polarization of monocytic EPCs.

OBJECTIVE Reduction of bone marrow–derived circulating progenitor cells has been proposed as a novel mechanism of cardiovascular disease in type 2 diabetes. The present study was designed to describe the extent and potential mechanisms of progenitor cell reduction during the natural history of type 2 diabetes. RESEARCH DESIGN AND METHODS We identified 425 individuals, divided into seven categories according to carbohydrate metabolism status (normal glucose tolerance [NGT], impaired fasting glucose, impaired glucose tolerance [IGT], and newly diagnosed type 2 diabetes) and diabetes duration (0–9, 10–19, and ≥20 years). These categories were examined as ideally describing the natural history of type 2 diabetes development and progression. We measured CD34+ and CD34+KDR+ progenitor cells by flow cytometry. We also evaluated progenitor cells in 20 coupled bone marrow and peripheral blood samples and examined progenitor cell apoptosis in 34 subjects. RESULTS In comparison to NGT, CD34+ cells were significantly reduced in IGT and had a first nadir in newly diagnosed type 2 diabetes and a second nadir after 20 years of diabetes. Statistical adjustment for possible confounders confirmed that CD34+ cell counts are deeply reduced at time of diagnosis, that they partially recover during the subsequent 0–19 years, and that they dip again after ≥20 years. A similar, but less consistent, trend was detected for CD34+KDR+ cells. Peripheral blood CD34+ cells were directly correlated with bone marrow CD34+ cells and inversely correlated with CD34+ cell apoptosis. CONCLUSIONS Circulating progenitor cell reduction marks the clinical onset of type 2 diabetes. Both defective mobilization and increased apoptosis may account for this phenomenon. While a partial recovery occurs during subsequent years, bone marrow reserve seems exhausted in the long term.

OBJECTIVE The redox enzyme p66Shc produces hydrogen peroxide and triggers proapoptotic signals. Genetic deletion of p66Shc prolongs life span and protects against oxidative stress. In the present study, we evaluated the role of p66Shc in an animal model of diabetic wound healing. RESEARCH DESIGN AND METHODS Skin wounds were created in wild-type (WT) and p66Shc−/− control and streptozotocin-induced diabetic mice with or without hind limb ischemia. Wounds were assessed for collagen content, thickness and vascularity of granulation tissue, apoptosis, reepithelialization, and expression of c-myc and β-catenin. Response to hind limb ischemia was also evaluated. RESULTS Diabetes delayed wound healing in WT mice with reduced granulation tissue thickness and vascularity, increased apoptosis, epithelial expression of c-myc, and nuclear localization of β-catenin. These nonhealing features were worsened by hind limb ischemia. Diabetes induced p66Shc expression and activation; wound healing was significantly faster in p66Shc−/− than in WT diabetic mice, with or without hind limb ischemia, at 1 and 3 months of diabetes duration and in both SV129 and C57BL/6 genetic backgrounds. Deletion of p66Shc reversed nonhealing features, with increased collagen content and granulation tissue thickness, and reduced apoptosis and expression of c-myc and β-catenin. p66Shc deletion improved response to hind limb ischemia in diabetic mice in terms of tissue damage, capillary density, and perfusion. Migration of p66Shc−/− dermal fibroblasts in vitro was significantly faster than WT fibroblasts under both high glucose and hypoxia. CONCLUSIONS p66Shc is involved in the delayed wound-healing process in the setting of diabetes and ischemia. Thus, p66Shc may represent a potential therapeutic target against this disabling diabetes complication.

Bone marrow-derived progenitor cells are involved in the homeostasis of the cardiovascular system through differentiation into endothelium, smooth muscle, and cardiomyocytes. Alterations of these extremely plastic cells have been recognized as both markers of cardiovascular risk and pathophysiological links between risk factors and development of atherosclerosis. Metabolic syndrome, as a cluster of well-defined cardiovascular risk factors, represents a strong predictor of cardiovascular events and death. Moreover, components of the syndrome interact with one another and synergistically increase this risk. Here we describe all metabolic syndrome components as being characterized by alterations in circulating progenitor cells, especially endothelial cells. We also highlight how endothelial progenitors may mediate the interactions between cardiometabolic risk factors in a complex interplay and discuss potential implications for prevention and therapy.

Introduction Primary antiphospholipid antibody syndrome (PAPS) is characterized by venous or arterial thrombosis and positive antiphospholipid antibodies. It is controversial whether PAPS patients have early atherosclerosis. Endothelial dysfunction is an early event in the natural history of atherosclerosis. Aim of our study was to compare endothelial function of patients with PAPS and no associated risk factors with that of age- and sex-matched controls. Materials and Methods Patients with PAPS, carefully selected to exclude all known risk factors for cardiovascular diseases, estrogen therapy, pregnancy, intake of drugs affecting endothelial function, vitamins or antioxidants, were included in a case-control study. Controls were age- (± 5 years) and sex-matched subjects with the same exclusion criteria but without PAPS. Flow-mediated dilation of the brachial artery and some plasmatic markers of endothelial and platelet activation were measured. Measures are expressed as mean±SEM. Results Twenty cases (mean age 42 ± 4.0 years, 11 females) and 39 controls (mean age 41 ± 2.9, 22 females) were studied. FMD was 5.7 ± 0.8% in cases (95% CI: 4.1 to 7.3) and 6.8 ± 0.5% (5.7 to 7.9) in controls (p = NS). Plasma von Willebrand factor was 128 ± 11.3% and 134.2 ± 16.1% in cases and controls, respectively (p = NS). Soluble P-selectin and soluble CD40L were 94.1 ± 4.9 ng/ml and 0.7 ± 0.1 ng/ml in cases and 87.7 ± 4.0 ng/ml and 1.0 ± 0.2 in controls, respectively (p = NS). In a substudy, circulating progenitor and mature endothelial cells were comparable between the two groups. Conclusions Endothelial function in patients with PAPS and no associated risk factors is similar to that of age- and sex- matched controls. These data suggest that the alterations leading to thrombosis in PAPS concern primarily the clotting system.

Hematopoietic stem cell transplantation (HSCT) is a curative therapy for a variety of hematologic malignancies. Recent studies have indicated the presence of donor-derived alloreactive NK cells as an additional factor influencing transplantation outcome, NK cells being regulated by signals delivered from multiple activating and inhibitory receptors. In the allogeneic HSCT setting, several studies considered donor-recipient HLA-I mismatch as the basis for NK alloreactivity. The implication of the KIR ligand incompatibility model is that donor’s NK cells exhibit the relevant inhibitory KIR for the self HLA-I ligands. It is known, however, that the KIR genes and the HLA genes segregate independently of each other in normal mendelian fashion. This allows the possibility that individuals lack HLA ligands for their KIR receptors and indicates that NK cells alloreactivity can occur also in HLA-matched or HLA-identical transplants. Recent investigations suggest that relapse and KIR-driven alloreactivity in the transplantation setting might be better predicted if the donor KIR genotype is considered in addition to the HLA genotype of the recipient. The aim of this study was to investigate at genetic level the prognostic impact of recipient HLA-I lacking for donor KIR on allotransplanted patients outcome. We analyzed donors KIR genotype and HLA genotype of 60 pediatric patients who received related (n.15) or unrelated (n.45) transplantation. When patients were grouped on the basis of the KIR gene type involved in the KIR/HLA-I mismatch, we did not observe any relapse in the group of patients characterized by mismatches involving only inhibitory KIR. On the contrary, all relapses were observed in patients showing at least one activating gene involved in the mismatch (p

Background NK cells represent a subset of lymphocytes belonging to the innate immunity branch typically expressing CD16 and CD56 associated to CD3 negativity. Two major subtypes of NK cells can be distinguished through CD16 and CD56 expression: CD56high/CD16dim/neg NK cells with low cytotoxic function and CD56dim/CD16high NK cells with high cytotoxic function. Recently a subtype of NK cells with memory properties characterized by CD56dim/CD16high/CD57+/CD62L- phenotype has been discovered. Chronic Lymphoproliferative Disorders of Granular Lymphocytes are characterized by the clonal expansion of Large Granular Lymphocyte (LGL) that can be CD3 positive (T-LGLL) or CD3 negative (Chronic Lymphoproliferative Disorder of NK Cell, CLPD-NK). The disease generally has indolent course but some patients develop cytopenia, particularly neutropenia, exposing to potentially lethal bacterial infections. Furthermore, NK-CLPD is usually referred as a more indolent disorder with respect to T-LGLL, with lower incidence of cytopenia and treatment need. CLPD-NK therapy does not differ from that of T-LGLL and is usually represented by an immunosuppressive therapy with low dose cyclophosphamide or methotrexate, with cyclosporine A usually being reserved to refractory patients. Somatic STAT3 mutations represent a new diagnostic marker of these disorders, initially reported in T-LGLL in about 40% oh patients, but also present in CLPD-NK in about 30% of cases. Using flow analysis, the aim of the present study was to identify a subset of CLPD NK patients characterized by a more severe disease requiring a shorter follow-up as compared to patients with a more indolent disease. Methods In a cohort of 16 patients affected by CLPD-NK, NK cells were analysed by flow for CD3, CD16, CD56, CD57 and CD62L antigen expression. These patients were studied for the presence of cytopenia and treatment requirement. STAT3 mutation analysis of exon 21 was performed with Sanger sequencing. Finally, p-STAT3 tyr 705 level and total STAT3 level were examined by western blotting. Results In relation to CD16 and CD56 expression, three major NK cells populations can be recognized in CLPD-NK patients: CD56high/CD16neg NK cells, CD56dim/CD16neg NK cells and CD56neg/dim/CD16high. As a consequence, patients can be separated into three groups characterized by the preferentially expansion of one of these populations: 2/16 (13%) with CD56high/CD16neg NK population, 4/16 (25%) with CD56dim/CD16neg NK population and 10/16 (62%) with CD56neg/dim/CD16high NK population. Furthermore, patients with predominance of this last NK cells subset were studied for CD57 and CD62L expression to identify NK cytotoxic subset (CD57-/CD62Llow/neg) and NK memory subset (CD57+/CD62Llow/neg); a NK cytotoxic/memory ratio (C/M ratio) was then calculated. 4 of 10 CD56neg/dim/CD16high patients (40%) were characterized by prevalence of NK cytotoxic cells expansion and high C/M ratio (≥3) while the remaining 6/10 patients were characterized by NK memory cells expansion with low C/M ratio (≤1.6). We then evaluated the presence of cytopenia, in particular neutropenia, in our patients' cohort. Neutropenia was shown in 7/16 (44%) patients with 4/16 (25%) experiencing severe neutropenia. Anemia and thrombocytopenia were less frequent (19% and 6% respectively). Interestingly, 6 out of 7 (86%) neutropenic patients were in the CD56neg/dim/CD16high subset and all patients with severe neutropenia belonged to the high C/M ratio subset. Interestingly, 3 out of 4 patients (75%) of this subset required therapy during the natural history of the disease. Concerning STAT3 mutation analysis, no one mutated patient was found in this setting. By western blot analysis, patients with high C/M ratio presented higher p-STAT3 levels than other patients and normal NK cells. Summary Although CLPD-NK represents an extreme heterogeneous disorder, discrete subtypes of disease characterized by different NK cells population expansion can be identified by flow analysis. Interestingly, this splitting allows to identify a subset of patients with prevalence of CD56neg/dim/CD16high NK cells with high C/M ratio that are characterized by high level of p-STAT3, high frequency of severe neutropenia and treatment requirement. Disclosures No relevant conflicts of interest to declare.

Introduction Several prognostic factors have been identified to predict the outcome of patients with chronic lymphocytic leukemia (CLL), but only a few studies analyzed more markers together. Patients and Methods Taking advantage of a population of 608 patients, we identified the strongest prognostic markers of survival and, subsequently, in a cohort of 212 patients we integrated data of cytogenetic lesions, IGHV mutational status, and CD38 expression in a new and easy scoring system we called the integrated CLL scoring system (ICSS). ICSS defines 3 groups of risk: (1) low risk (patients with 13q − or normal fluorescence in-situ hybridization analysis results, mutated IGHV, and CD38) (2) high risk (all 11q − or 17p − patients and/or all unmutated IGHV and CD38 + patients); and (3) intermediate risk (all remaining patients). Results Using only these 3 already available prognostic factors, we were able to properly redefine patients and better predict the clinical course of the disease. Conclusion ICSS could become a useful tool for CLL patients' management.

By analyzing the expression of several cytotoxic markers, killer-immunoglobulin-like receptors (KIRs), CD94/CD159, CD314 and natural cytotoxicity receptors (NCRs), in 22 CD3+ lymphoproliferative disease of granular lymphocyte (LDGL) patients we investigated whether granular lymphocytes (GLs) displayed the phenotype of fully differentiated cytotoxic cells. Our results demonstrate that GLs express a pattern consistent with fully differentiated CTLs. KIRs are expressed only in a fraction of patients (7/22), as is CD94/CD159 (5/22). In conclusion, GLs in CD3+ LDGL patients typically show the phenotype of fully differentiated CTL, whereas the expression of NK receptors does not represent a common feature of the proliferating clone.

T large granular lymphocytes leukemia (T-LGLL) and NK-type chronic lymphoproliferative disorder (CLPD-NK) are rare diseases characterized by the abnormal expansion of large granular lymphocytes (LGLs) with cytotoxic activity, belonging to T and NK lineage, respectively. Currently, the etiology of these diseases is still largely unknown. Several data support the hypothesis that the inciting event is represented by the persistence of antigenic stimulation, maintained by the abnormal release of cytokines (mainly IL-6 and IL-15), establishing an inflammation status not achieving resolution. Recently, we showed that IL-6 and soluble IL-6Rα were highly expressed and released by patients’ LGL-depleted peripheral blood mononuclear cells (PBMC), accounting for a trans-signaling process. IL-6 trans-signaling is critically involved in inflammatory disease and promotes the transition from acute to chronic inflammation. Additionally, LGL proliferation is maintained for an impairment of the apoptotic machinery due to the activation of many survival signaling pathways, including JAK/STAT and RAS/MEK/ERK pathways. In some patients (both T-LGLL and CLPD-NK) STAT3 hot-spot mutations, inducing STAT3 activation, have been demonstrated. With this as a background, we investigated the IL-15 contribution to sustain IL-6 trans-signaling and in turn inflammation. We analyzed the relationships between STAT3 mutations, IL-6 and IL-15 in disease progression to assess the hypothesis that these findings characterize different stages of LGL disease. Thirty T-LGLL and 15 CLPD-NK patients were included in this study. Patients were subdivided according to the percentage of LGLs in PBMCs (LGL range: 35-90%). By ELISA in patients’ plasma, we showed that IL-6 concentrations were significantly higher in patients characterized by a disease with less than 60% circulating LGLs (35.7 ± 11.4 pg/ml with respect to patients with LGLs >60%: 9.1 ± 2.7 pg/ml; p These results suggest that an initial step along the development of disease characterized by a low lymphocytosis (LGLs 60% of total PBMCs) in which LGL disease goes on independently from exogenous stimuli, likely becoming self-maintaining due to the contribution of the emerging STAT3 mutations. It is suggested that these phases can represent two sequential steps in the progression of disease. Disclosures: No relevant conflicts of interest to declare.