Samuela Carraro

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.

Hyperforin down-regulates effector function of activated T lymphocytes and shows efficacy against Th1-triggered CNS inflammatory-demyelinating disease

Hyperforin (Hyp) is an active compound contained in the extract of Hypericum perforatum, well known for its antidepressant activity. However, Hyp has been found to possess several other biological properties, including inhibitory effects on tumor invasion, angiogenesis, and inflammation. In this paper, we show that treatment with Hyp inhibited IFN‐γ production, with down‐regulation of T‐box (T‐bet; marker of Th1 gene expression) and up‐regulation of GATA‐3 (marker gene of Th2) on IL‐2/PHA‐activated T cells. In parallel, we showed a strong down‐regulation of the chemokine receptor CXCR3 expression on activated T cells. The latter effect and the down‐modulation of matrix metalloproteinase 9 expression may eventually lead to the inhibition of migratory capability and matrix traversal toward the chemoattractant CXCL10 by activated lymphocytes that we observed in vitro. The effect of Hyp was thus evaluated on an animal model of experimental allergic encephalomyelitis (EAE), a classic, Th1‐mediated autoimmune disease of the CNS, and we observed that Hyp attenuates the severity of the disease symptoms significantly. Together, these properties qualify Hyp as a putative, therapeutic molecule for the treatment of autoimmune inflammatory disease sustained by Th1 cells, including EAE.

HS1, a Lyn Kinase Substrate, Is Abnormally Expressed in B-Chronic Lymphocytic Leukemia and Correlates with Response to Fludarabine-Based Regimen

In B-Chronic Lymphocytic Leukemia (B-CLL) kinase Lyn is overexpressed, active, abnormally distributed, and part of a cytosolic complex involving hematopoietic lineage cell-specific protein 1 (HS1). These aberrant properties of Lyn could partially explain leukemic cells’ defective apoptosis, directly or through its substrates, for example, HS1 that has been associated to apoptosis in different cell types. To verify the hypothesis of HS1 involvement in Lyn-mediated leukemic cell survival, we investigated HS1 protein in 71 untreated B-CLL patients and 26 healthy controls. We found HS1 overexpressed in leukemic as compared to normal B lymphocytes (1.38±0.54 vs 0.86±0.29, p<0.01), and when HS1 levels were correlated to clinical parameters we found a higher expression of HS1 in poor-prognosis patients. Moreover, HS1 levels significantly decreased in ex vivo leukemic cells of patients responding to a fludarabine-containing regimen. We also observed that HS1 is partially localized in the nucleus of neoplastic B cells. All these data add new information on HS1 study, hypothesizing a pivotal role of HS1 in Lyn-mediated modulation of leukemic cells’ survival and focusing, one more time, the attention on the BCR-Lyn axis as a putative target for new therapeutic strategies in this disorder.

TL1A/DR3 axis involvement in the inflammatory cytokine network during pulmonary sarcoidosis

BackgroundTNF-like ligand 1A (TL1A), a recently recognized member of the TNF superfamily, and its death domain receptor 3 (DR3), firstly identified for their relevant role in T lymphocyte homeostasis, are now well-known mediators of several immune-inflammatory diseases, ranging from rheumatoid arthritis to inflammatory bowel diseases to psoriasis, whereas no data are available on their involvement in sarcoidosis, a multisystemic granulomatous disease where a deregulated T helper (Th)1/Th17 response takes place.MethodsIn this study, by flow cytometry, real-time PCR, confocal microscopy and immunohistochemistry analyses, TL1A and DR3 were investigated in the pulmonary cells and the peripheral blood of 43 patients affected by sarcoidosis in different phases of the disease (29 patients with active sarcoidosis, 14 with the inactive form) and in 8 control subjects.ResultsOur results demonstrated a significant higher expression, both at protein and mRNA levels, of TL1A and DR3 in pulmonary T cells and alveolar macrophages of patients with active sarcoidosis as compared to patients with the inactive form of the disease and to controls. In patients with sarcoidosis TL1A was strongly more expressed in the lung than the blood, i.e., at the site of the involved organ. Additionally, zymography assays showed that TL1A is able to increase the production of matrix metalloproteinase 9 by sarcoid alveolar macrophages characterized, in patients with the active form of the disease, by reduced mRNA levels of the tissue inhibitor of metalloproteinase (TIMP)-1.ConclusionsThese data suggest that TL1A/DR3 interactions are part of the extended and complex immune-inflammatory network that characterizes sarcoidosis during its active phase and may contribute to the pathogenesis and to the progression of the disease.

Detection of monoclonal T populations in patients with KIR-restricted chronic lymphoproliferative disorder of NK cells

The etiology of chronic large granular lymphocyte proliferations is largely unknown. Although these disorders are characterized by the expansion of different cell types (T and natural killer) with specific genetic features and abnormalities, several lines of evidence suggest a common pathogenetic mechanism. According to this interpretation, we speculated that in patients with natural killer-type chronic lymphoproliferative disorder, together with natural killer cells, also T lymphocytes undergo a persistent antigenic pressure, possibly resulting in an ultimate clonal T-cell selection. To strengthen this hypothesis, we evaluated whether clonal T-cell populations were detectable in 48 patients with killer immunoglobulin-like receptor-restricted natural killer-type chronic lymphoproliferative disorder. At diagnosis, in half of the patients studied, we found a clearly defined clonal T-cell population, despite the fact that all cases presented with a well-characterized natural killer disorder. Follow-up analysis confirmed that the TCR gamma rearrangements were stable over the time period evaluated; furthermore, in 7 patients we demonstrated the appearance of a clonal T subset that progressively matures, leading to a switch between killer immunoglobulin-like receptor-restricted natural killer-type disorder to a monoclonal T-cell large granular lymphocytic leukemia. Our results support the hypothesis that a common mechanism is involved in the pathogenesis of these disorders.

CXCR6-CXCL16 interaction in the pathogenesis of Juvenile Idiopathic Arthritis ☆

In order to evaluate the role of CXCR6/CXCL16 in driving lymphocyte migration into inflamed joints of children with oligoarticular Juvenile Idiopathic Arthritis (JIA) we analysed CXCR6 expression and functional capability in lymphocytes from synovial fluid (SF) by flow cytometry, by real-time polymerase chain reaction (RT-PCR) and migration assays. Furthermore, CXCR6 and CXCL16 expression in synovial tissue (ST) was analysed by immunohistochemistry. T cells isolated from SF of patients with JIA expressed CXCR6 which was functionally active as shown by chemotactic assays. The same cells expressed CXCR3 and it exerted a migratory activity in response to CXCL10. CXCL16 and CXCR6 were intensively expressed on the synovium cells, respectively on macrophages, synoviocytes and endothelial cells and on lymphocytes, synoviocytes and endothelial cells. Taken together, these data suggest that CXCR6 and CXCR3 act coordinately with respective ligands and are involved in the pathophysiology of JIA-associated inflammatory processes.

Dominant cytotoxic NK cell subset within CLPD-NK patients identifies a more aggressive NK cell proliferation

Natural killer (NK) cells represent a class of innate lymphocytes with large granular morphology and cytotoxic functions, characterized by the CD3–/CD16+/CD56 + phenotype. According to CD56 expression, two major NK cell subsets can be recognized, CD56/CD16 NK cells with the ability to release cytokines and CD56/CD16 NK cells displaying cytotoxic ability toward virus infected or neoplastic cells. NK cells are traditionally considered part of innate immunity but evidence has been recently provided that a distinct NK cell subset may respond to specific antigens like adaptive immune cells. These NK cells, also known as “NK memory”, are induced by the chronic stimulation of viral infections or by cytokines (IL-12, IL-15 and IL-18) and are included in the CD56/CD16 NK cells subgroup equipped with CD57 but lacking CD62L. Chronic lymphoproliferative disorder of NK cells (CLPD-NK) is a provisional entity, recognized by the 2016 WHO classification, characterized by chronic expansion of at least 500/ mm NK cells with restricted killer immunoglobulin-like receptor (KIR) pattern, whose assessment is of crucial relevance due to the lack of T-cell receptor rearrangement in NK cells. CLPD-NK has generally an indolent course, most patients being asymptomatic and the main feature of the disease being represented by the development of neutropenia. Only few data are available on the pathogenesis of this indolent disorder but a constitutive activation of anti-apoptotic signaling pathways is likely to play a relevant role. The discovery in 2012 of somatic STAT3 mutations, in about 40% of T-cell large granular lymphocyte leukemia (T-LGLL) and in about 30% of CLPDNK, focused the attention on a constitutive activation of Janus Kinase-Signal Transducers and Activators of Transcription (JAK-STAT) pathway in the development of this disorder. In addition, somatic STAT5bmutations were recently recognized in rare aggressive variants of LGLL and indolent CD4+ T-LGLL. This rare disorder remains an extremely heterogeneous disease, some patients being completely asymptomatic, whereas others require specific treatment. For this reason, using flow cytometry and regardless of KIR expression, the aim of this study was to identify different biological and clinical CLPD-NK patients’ subsets. By flow analysis, NK cells of 25 patients affected by CLPD-NK were analyzed for CD16 and CD56 expression (Supplementary Methods), recognizing two major NK cell subsets, that is, patients with CD56/CD16 NK cells (4/25, 16%) and patients with CD56/CD16 NK cells (21/25, 84%) (Figs. 1a–c). All patients were evaluated for clinical and hematological characteristics. Median age was 62 years (range 42–79) with no significant difference between CD56/CD16 and CD56/CD16 subgroups. As expected, neutropenia (absolute neutrophil count (ANC) < 1500/mm) was the most relevant feature, detected in 10 out of 25 patients (40%), with 5 patients (20%) presenting severe neutropenia (ANC < 500/mm). Anemia and thrombocytopenia have been detected only in a minority of patients (2/25 and 3/25, respectively) and were generally mild (Table 1). In terms of clinical presentation, almost all symptomatic patients were included in the CD56/CD16 subgroup with 8 out of 21