Lack of expression of inhibitory KIR3DL1 receptor in patients with natural killer cell-type lymphoproliferative disease of granular lymphocytes.

BACKGROUND: Natural killer cell-type lymphoproliferative disease of granular lymphocytes is a disorder characterized by chronic proliferation of CD3(-)CD16(+) granular lymphocytes. By flow cytometry analysis, we previously demonstrated a dysregulation in killer immunoglobulin-like receptor (KIR) expression in natural killer cells from patients with this lymphoproliferative disease, the activating KIR receptors being mostly expressed. We also found that patients with natural killer cell-type lymphoproliferative disease of granular lymphocytes usually had KIR genotypes characterized by multiple activating KIR genes. DESIGN AND METHODS: We investigated the mRNA levels of the KIR3DL1 inhibitory and the related KIR3DS1 activating receptors in 15 patients with natural killer cell-type lymphoproliferative disease of granular lymphocytes and in ten controls. These genes are usually expressed when present in the genome of the Caucasian population. RESULTS: We demonstrated the complete lack of KIR3DL1 expression in most of the patients analyzed, with the receptor being expressed in 13% of patients compared to in 90% of controls (P<0.01). Interestingly, studies of the methylation patterns of KIR3DL1 promoter showed a significantly higher methylation status (0.76 ± 0.12 SD) in patients than in healthy subjects (0.49±0.10 SD, P<0.01). The levels of expression of DNA methyl transferases, which are the enzymes responsible for DNA methylation, did not differ between patients and controls. CONCLUSIONS: In this study we showed, for the first time, a consistent down-regulation of the inhibitory KIR3DL1 signal due to marked methylation of its promoter, thus suggesting that together with the increased expression of activating receptors, the lack of the inhibitory signal could also play a role in the pathogenesis of natural killer cell-type lymphoproliferative disease of granular lymphocytes.

A Multi-Center, Real-Life Experience on Liquid Biopsy Practice for EGFR Testing in Non-Small Cell Lung Cancer (NSCLC) Patients.

BACKGROUND: circulating tumor DNA (ctDNA) is a source of tumor genetic material for EGFR testing in NSCLC. Real-word data about liquid biopsy (LB) clinical practice are lacking. The aim of the study was to describe the LB practice for EGFR detection in North Eastern Italy. METHODS: we conducted a multi-regional survey on ctDNA testing practices in lung cancer patients. RESULTS: Median time from blood collection to plasma separation was 50 min (20-120 min), median time from plasma extraction to ctDNA analysis was 24 h (30 min-5 days) and median turnaround time was 24 h (6 h-5 days). Four hundred and seventy five patients and 654 samples were tested. One hundred and ninety-two patients were tested at diagnosis, with 16% EGFR mutation rate. Among the 283 patients tested at disease progression, 35% were T790M+. Main differences in LB results between 2017 and 2018 were the number of LBs performed for each patient at disease progression (2.88 vs. 1.2, respectively) and the percentage of T790M+ patients (61% vs. 26%).

Chronic lymphocytic leukemia B cells contain anomalous Lyn tyrosine kinase, a putative contribution to defective apoptosis.

B cell chronic lymphocytic leukemia (B-CLL) is a neoplastic disorder characterized by accumulation of B lymphocytes due to uncontrolled growth and resistance to apoptosis. Analysis of B cells freshly isolated from 40 patients with chronic lymphocytic leukemia demonstrated that the Src kinase Lyn, the switch molecule that couples the B cell receptor to downstream signaling, displays anomalous properties. Lyn is remarkably overexpressed at the protein level in leukemic cells as compared with normal B lymphocytes, with a substantial aliquot of the kinase anomalously present in the cytosol. Whereas in normal B lymphocytes Lyn activation is dependent on B cell-receptor stimulation, in resting malignant cells, the constitutive activity of the kinase accounts for high basal protein tyrosine phosphorylation and low responsiveness to IgM ligation. Addition of the Lyn inhibitors PP2 and SU6656 to leukemic cell cultures restores cell apoptosis, and treatment of malignant cells with drugs that induce cell apoptosis decreases both activity and amount of the tyrosine kinase. These findings suggest a direct correlation between high basal Lyn activity and defects in the induction of apoptosis in leukemic cells. They also support a critical role for Lyn in B-CLL pathogenesis and identify this tyrosine kinase as a potential therapeutic target.

Expression and role of CCR6/CCL20 chemokine axis in pulmonary sarcoidosis.

We have shown previously that the chemokine receptors CXCR3 and CXCR6 are coexpressed by Th1 cells infiltrating the lung and the granuloma of patients with sarcoidosis. In this study, we evaluated the role of CCL20/CCR6 interaction in the pathogenesis of acute and chronic pulmonary sarcoidosis. By flow cytometry and molecular analyses, we have demonstrated that Th1 cells isolated from the bronchoalveolar lavage (BAL) of patients with sarcoidosis and T cell alveolitis are equipped with CCR6. Furthermore, CCR6(+) T cells coexpressed the chemokine receptors 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 macrophage (AM) surface, and it is observed in the cytoplasm 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. Furthermore, 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 assays. In vitro kinetic studies demonstrated that CCR6 is induced rapidly by IL-2, IL-18, and IFN-gamma. In conclusion, T cells expressing CCR6, CXCR3, and CXCR6 act coordinately with respective ligands and Th1 inflammatory cytokines in the alveolitic/granuloma phases of the disease.

T-cell type lymphoproliferative disease of granular lymphocytes (LDGL) is equipped with a phenotypic pattern typical of effector cytotoxic cells.

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.

Circulating endothelial progenitor cells are reduced in peripheral vascular complications of type 2 diabetes mellitus.

OBJECTIVES: We sought to establish whether a reduction in endothelial progenitor cells (EPCs) has a putative role in peripheral vascular disease (PVD) of type 2 diabetic patients. BACKGROUND: Peripheral vascular disease is a common and severe complication of diabetes mellitus. Impaired collateralization of diabetic vasculopathy has been extensively shown, but causes leading to its pathogenesis are not fully understood. Recently, EPCs have been found to contribute to vascular repair and angiogenesis. Diabetes has been associated with low levels of circulating EPCs, but no data are available in the literature on the relationship between EPCs and PVD in diabetes. METHODS: Flow cytometric analysis was used to quantify circulating progenitor cells (CPCs, CD34+) and EPCs (CD34+KDR+) in 51 patients and 17 control subjects. RESULTS: The CPCs and EPCs from diabetic patients were reduced by 33% and 40%, respectively, compared with healthy subjects (p < 0.001). An inverse correlation was found between the number of EPCs and the values of fasting glucose (r = -0.49, p = 0.006). Peripheral vascular disease was associated with a 47% reduction in EPCs (p < 0.0001) and EPC levels directly correlated with the ankle-brachial index (r = 0.70, p = 0.01). The subgroup of diabetic patients with PVD also had reduced CPCs by 32% (p = 0.037), whereas patients with ischemic foot lesions had the lowest levels of both EPCs and CPCs (p = 0.02). CONCLUSIONS: Our data demonstrate decreased EPC levels in diabetic patients and, for the first time, show that PVD is associated with an extensively low number of EPCs. Depletion of circulating EPCs in diabetic patients may be involved in the pathogenesis of peripheral vascular complications.

HCV-related liver and lymphoproliferative diseases: association with polymorphisms of IL28B and TLR2.

To explore the relationship between innate immunity and hepatitis C Virus (HCV) in determining the risk of cirrhosis (CIR), hepatocellular carcinoma (HCC), mixed cryoglobulinemia syndrome (MCS) and non-Hodgkin lymphoma (NHL), we investigated the impact of the toll-like receptor-2 (TLR2) and interleukin-28B (IL28B) genetic variants. TLR2 -174 del variant was associated with TLR2 expression and with specific downstream molecules that drive the expression of different interleukins; rs12979860 Il28B was important in response to interferon-treatment and in spontaneous clearance of HCV. The risk for liver and lymphoproliferative diseases in HCV progression was clarified by stratifying 862 HCV-positive patients into groups based on liver (CIR, HCC) and lymphoproliferative HCV-related diseases (MCS, NHL) and compared with chronic HCV (CHC) infection. Analysis of TLR2-IL28B haplotypes showed an association of wild type haplotype with the lymphoproliferative diseases (OR 1.77, p = 0.029) and a slight increase in HCV viral load (HR 1.38, p = 0.054). Wild type haplotype (TLR2 ins/ins- IL28B C/C) was also found associated with older age in patients with an hepatic diseases (in CIR and in HCC p = 0.038 and p = 0.020, respectively) supporting an effect of innate immunity in the liver disease progression. TLR2 and IL28B polymorphisms in combination showed a role in the control of HCV viral load and different HCV disease progression.

Arterio-venous gradient of endothelial progenitor cells across renal artery stenosis.

A role for circulating progenitor cells have been recently demonstrated in many pathological conditions. Endothelial progenitor cells (EPCs) localize at sites of ischemia and stimulate neovasculogenesis. This small study was carried out to assess whether selective blood sampling during renal angiography could demonstrate an arterio-venous gradient of EPCs in 5 patients with renal artery stenosis. Surprisingly, we found that EPCs were more abundant in venous than in arterial blood, suggesting that the kidney subjected to chronic ischemia may become a source rather than a target of EPCs.

CXCR3/CXCL10 interactions in the development of hypersensitivity pneumonitis.

BACKGROUND: Hypersensitivity pneumonitis (HP) is an interstitial lung disease caused by repeated inhalations of finely dispersed organic particles or low molecular weight chemicals. The disease is characterized by an alveolitis sustained by CD8(+) cytotoxic T lymphocytes, granuloma formation, and, whenever antigenic exposition continues, fibrosis. Although it is known that T-cell migration into the lungs is crucial in HP reaction, mechanisms implicated in this process remain undefined. METHODS: Using flow cytometry, immunohistochemistry, confocal microscopy analysis and chemotaxis assays we evaluated whether CXCL10 and its receptor CXCR3 regulate the trafficking of CD8(+) T cells in HP lung. RESULTS: Our data demonstrated that lymphocytes infiltrating lung biopsies are CD8 T cells which strongly stain for CXCR3. However, T cells accumulating in the BAL of HP were CXCR3(+)/IFNgamma(+) Tc1 cells exhibiting a strong in vitro migratory capability in response to CXCL10. Alveolar macrophages expressed and secreted, in response to IFN-gamma, definite levels of CXCL10 capable of inducing chemotaxis of the CXCR3(+) T-cell line. Interestingly, striking levels of CXCR3 ligands could be demonstrated in the fluid component of the BAL in individuals with HP. CONCLUSION: These data indicate that IFN-gamma mediates the recruitment of lymphocytes into the lung via production of the chemokine CXCL10, resulting in Tc1-cell alveolitis and granuloma formation.

T cells in the lung of patients with hypersensitivity pneumonitis accumulate in a clonal manner.

Hypersensitivity pneumonitis (HP) is characterized by an alveolitis sustained by CD8(+) T lymphocytes showing a limited expression of the T cell receptor (TCR). We previously demonstrated that a bias in T cell selection occurs in the lower respiratory tract of patients with HP, with a compartmentalization in the lung of CD8(+) T cells bearing (TCR)-beta variable (TCRBV) #2, 3, 5, 6, 8, and 13 gene segments. We herein characterized the clonal T cell populations present in the lung and in the blood of patients with HP. Heteroduplex analyses, cloning, and sequencing T cells bearing TCR indicate oligoclonal expansions of T cells expressing homologous or identical complementary-determining region 3. Furthermore, T cell clones isolated from the two compartments expressed similar, sometimes identical, junctional regions. Removal from antigenic exposure led to the disappearance of T cell clones. Our findings indicate that expansions of T lymphocytes bearing clonal TCRBV region gene segments take place in the lung of patients with HP during exposure. The evidence that identical T cell clones are present in the lung and the blood of the same patient suggests that the immune reaction occurring at lung level gives rise to a systemic reaction.

Gene expression profile analysis by DNA microarrays: a new approach to assess functional genomics in diseases.

A biologically based and potentially powerful way to characterize human diseases has arisen from the new science of genomics. One of the methodologies to provide a systematic way to observe and characterize gene expression programs is the DNA microarrays (also called gene chips). This modern genomic analysis tool shows the promise of providing an unbiased approach to detecting biologically relevant differences within clinically similar diseases. Taking advantage of the initial reports that appeared in the literature on this topic, the likely impact of a molecular taxonomic of interstitial lung disease subgroups based upon gene expression profiling is discussed.

Multiple myeloma plasma cells show different chemokine receptor profiles at sites of disease activity.

Chemokines and their receptors play a pivotal role in the regulation of B-lymphocyte trafficking. This study was aimed at investigating the pattern of chemokine receptor expression, including CCR1 to CCR3, CCR5 to CCR7, CXCR1 to CXCR5, and the migration ability of multiple myeloma (MM) plasma cells (PC). PC were recovered from the bone marrow (BM) of 29 MM patients, extramedullary sites of 10 patients and the BM of five controls. Flow cytometry analysis showed that the receptors mainly expressed on malignant BM PC were represented by CXCR4 (70% of patients), CCR1 (25%), CCR2 (25%), CCR5 (17%) and CXCR3 (20%), while other receptors were commonly lacking. The analysis performed on extramedullary (peripheral blood and pleural effusion) malignant PC demonstrated that the most represented receptors were CXCR4 (100%), CCR2 (66%) and CXCR1 (60%). The migratory capability of malignant PC at resting conditions identified three groups of patients with different migration (low, intermediate and high). As CXCR4 was the relevant chemokine receptor expressed by MM PC, its ligand CXCL12 induced their migration. These data suggest that malignant PC from MM display different chemokine receptor profiles and that CXCR4 is fully functional and might play a role in the spreading of the disease.

Circulating progenitor cells are reduced in patients with severe lung disease.

Patients with chronic severe lung disease are prone to develop pulmonary vascular remodeling, possibly through pulmonary endothelial dysfunction. Circulating endothelial progenitor cells (EPCs) are involved in maintenance of endothelial homeostasis. The aim of this study was to assess whether obstructive and restrictive lung diseases are associated with modification of EPC number in peripheral blood. The study was cross-sectional and involved patients with obstructive (n = 15) and restrictive (n = 15) lung disease on oxygen therapy and 15 control subjects. Circulating EPCs were defined by the surface expression of CD34, CD133, and kinase-insert domain receptor. Results from spirometric tests, blood gas analyses, and blood cell counts have been related to EPC numbers. Patients with chronic hypoxia and severe lung disease showed lower levels of all progenitors than do control subjects. A consensual further reduction of EPC was found in restrictive patients in comparison with obstructive patients. Among restrictive patients, EPC reduction was related to reduced lung volumes and impaired alveolo-arterial diffusion, whereas progenitor cell levels were directly related to erythrocyte number. Considering obstructive patients, significant correlations were found between progenitor cell levels and bronchial obstruction and between progenitor cell levels and arterial oxygen tension. These findings demonstrate a reduction of EPCs in patients with chronic lung disease and long-lasting hypoxia. This alteration was more evident in restrictive patients and correlated to disease severity. Depletion of circulating EPCs may be involved in altered endothelial homeostasis of pulmonary circulation in these disorders.

Epithelial CXCR3-B regulates chemokines bioavailability in normal, but not in Sjogren's syndrome, salivary glands.

Expression of CXCR3-targeting chemokines have been demonstrated in several diseases, suggesting a critical role for CXCR3 in recruiting activated T cells to sites of immune-mediated inflammation. Sjögren's syndrome (SS) is an autoimmune disease characterized by a mononuclear cell infiltrate of activated T cells around the duct in the salivary gland. Analysis of minor salivary gland biopsy specimens from 20 healthy subjects and 18 patients with primary SS demonstrated that CXCR3, in particular, the B form of this receptor, is constitutively expressed by human salivary gland epithelial cells. Salivary gland epithelial cell cultures demonstrated that CXCR3 participate in removing relevant amount of agonists from the supernatant of exposed cells without mediating calcium flux or chemotaxis while retaining the ability to undergo internalization. Although in normal salivary gland epithelial cells, CXCR3 behaves as a chemokine-scavenging receptor, its role in SS cells is functionally impaired. The impairment of this scavenging function might favor chemotaxis, leading to heightened immigration of CXCR3-positive T lymphocytes. These findings suggest that epithelial CXCR3 may be involved in postsecretion regulation of chemokine bioavailability. They also support a critical role for CXCR3 in the pathogenesis of SS and identify its agonists as potential therapeutic targets.

Role for CXCR6 and its ligand CXCL16 in the pathogenesis of T-cell alveolitis in sarcoidosis.

RATIONALE: Receptor expression dictates the spectrum of chemokine actions on immunocompetent cells. We have previously shown that the chemokine receptor CXCR3 is highly expressed by T-helper type 1 (Th1) cells infiltrating the lungs of patients with sarcoidosis. OBJECTIVES: The evaluation of the role of Bonzo/CXCR6 and its ligand CXCL16 in the pathogenesis of sarcoidosis. METHODS: Immunocompetent cells infiltrating sarcoid lung have been evaluated by flow cytometry, confocal microscopy, immunohistochemical and molecular analysis, and functional assays. MAIN RESULTS: Th1 cells isolated from the bronchoalveolar lavage of patients with sarcoidosis and T-cell alveolitis coexpressed CXCR3 and CXCR6. Immunohistochemical analysis of lung specimens has shown that CXCR6+ T cells infiltrated lung interstitium surrounding the central core of the granuloma. The CXCR6 ligand CXCL16 was abundantly expressed by macrophages infiltrating sarcoid tissue and/or forming the granuloma core. From a functional point of view, sarcoid Th1 cells were able to respond to CXCL10 and CXCL16 in migratory assay. In vitro kinetic studies demonstrated that, although CXCR3 was rapidly induced by interleukin (IL)-15 and IL-18, CXCR6 induction was slow (8 d) and mainly regulated by IL-15. CONCLUSIONS: T cells coexpressing CXCR3 and CXCR6 act coordinately with respective ligands and Th1 inflammatory cytokines in the alveolitic/granuloma phases of the disease.

Phenotypic and functional analyses of dendritic cells in patients with lymphoproliferative disease of granular lymphocytes (LDGL).

We investigated whether dendritic cells (DCs) play a role in favoring granular lymphocyte (GL) proliferation in patients with lymphoproliferative disease of granular lymphocytes (LDGL). The presence of in vivo circulating DCs was studied in 11 patients (5 CD3+ and 6 CD3- LDGL). Autologous immature (iDCs) and mature (mDCs) DCs generated in vitro were studied for stimulatory activity on cell proliferation of CD3+ and CD3- GLs. The topographic organization of GLs and DCs was also studied in bone marrow (BM) biopsies. Peripheral blood (PB) CD3- GLs from patients showed significant proliferative activity in the presence of iDCs and mDCs. Conversely, monoclonal CD3+ GLs were unresponsive to autologous and allogeneic PB DCs. Analysis of BM biopsies demonstrated a topographic distribution of DCs and GLs that indicates contact between the 2 cell types. On functional assays, DCs obtained from BM were more efficient than PB DCs in stimulating CD3- GLs, and surprisingly, a low but definite stimulatory effect was demonstrated also on CD3+ GLs. The putative contact between DCs and GLs in the BM and, more crucial, the proliferative response of discrete GL populations to DC stimulation suggest the presence of a specific antigen within BM DCs, providing evidence for a role of DCs in the pathogenesis of LDGL.

Homeostatic chemokines drive migration of malignant B cells in patients with non-Hodgkin lymphomas.

This study investigated the role of several chemokines and their receptors on malignant B lymphocytes recovered from 13 patients with chronic lymphocytic leukemia (CLL), 9 with hairy cell leukemia (HCL), 5 with mantle cell lymphoma (MCL), 5 with marginal zone B-cell lymphoma (MZL), 6 with small lymphocytic lymphoma (SLL), and 5 with follicular cell lymphoma (FCL). Flow cytometry analysis demonstrated that CXCR4 and CXCR5 were expressed on all malignant and normal B cells. Considering CC receptors, CCR1 was expressed in 70% of patients with CLL and 40% of those with HCL but was lacking in patients with MCL, MZL, SLL, and normal B cells. CCR2 showed a heterogeneous pattern of expression. CCR3 was found in almost all patients with CLL and in the majority of those with HCL, whereas it was usually lacking in patients with MZL and SLL and in healthy subjects. CCR5 was expressed in patients with HCL and MCL. Migration assays showed that different chemokines, mainly CXCL12 and CXCL13, are able to trigger migration of malignant B lymphocytes. Some of these chemokines induce calcium mobilization. These data indicate that different patterns of chemokine receptor expression identify different malignant B-cell subsets and that these receptors are functional and might play a role in malignant B-cell circulation.