Elisa Ferretti

CCL19 and CXCL12 Trigger in Vitro Chemotaxis of Human Mantle Cell Lymphoma B Cells

Purpose: Few data are available in the literature on chemokine receptor expression and migratory capability of mantle cell lymphoma (MCL) B cells. Information on these issues may allow us to identify novel mechanisms of chemokine-driven tumor cell migration. Experimental Design: The research was designed to investigate: (a) expression of CCR1 to CCR7 and CXCR1 to CXCR5 chemokine receptors; and (b) chemotaxis to the respective ligands in MCL B cells and in their normal counterparts, i.e., CD5+ B cells. Results: Malignant B cells from MCL patients and normal counterparts displayed similar chemokine receptor profiles. MCL B cells were induced to migrate by CXCL12 and CCL19, whereas normal CD5+ B cells migrated to the former, but not the latter chemokine. Overnight culture of MCL B cells and their normal counterparts with CXCL12 cross-sensitized other chemokine receptors to their ligands in some tumor samples but not in CD5+ B cells. Conclusions: CCR7 and CXCR4 ligands may play a key role in tumor cell migration and spreading in vivo. CXCL12 may additionally contribute by sensitizing MCL B cells to respond to the ligands of other chemokine receptors.

Interleukin-27 Inhibits the Growth of Pediatric Acute Myeloid Leukemia in NOD/SCID/Il2rg−/− Mice

Purpose: Acute myeloid leukemia (AML) accounts for more than half of fatal cases in all pediatric leukemia patients; this observation highlights the need of more effective therapies. Thus, we investigated whether interleukin (IL)-27, an immunomodulatory cytokine, functions as an antitumor agent against pediatric AML cells. Experimental Design: Expression of WSX-1 and gp130 on AML cells from 16 pediatric patients was studied by flow cytometry. Modulation of leukemia cell proliferation or apoptosis upon IL-27 treatment in vitro was tested by bromodeoxyuridine/propidium iodide (PI) and Ki67, or Annexin V/PI staining and flow cytometric analysis. The angiogenic potential of AML cells treated or not with IL-27 was studied by chorioallantoic membrane assay and PCR array. In vivo studies were carried out using nonobese diabetic/severe combined immunodeficient (NOD/SCID)/Il2rg−/− mice injected intravenously with five pediatric AML cell samples. Leukemic cells engrafted in PBS and IL-27–treated animals were studied by immunohistochemical/morphologic analysis and by PCR array for expression angiogenic/dissemination-related genes. Results: We provided the first demonstration that (i) AML cells injected into NOD/SCID/Il2rg−/− mice gave rise to leukemia dissemination that was severely hampered by IL-27, (ii) compared with controls, leukemia cells harvested from IL-27–treated mice showed significant reduction of their angiogenic and spreading related genes, and (iii) similarly to what was observed in vivo, IL-27 reduced in vitro AML cell proliferation and modulated the expression of different genes involved in the angiogenic/spreading process. Conclusion: These results provide an experimental rationale for the development of future clinical trials aimed at evaluating the toxicity and efficacy of IL-27. Clin Cancer Res; 18(6); 1630–40. ©2012 AACR.

Soluble HLA-G dampens CD94/NKG2A expression and function and differentially modulates chemotaxis and cytokine and chemokine secretion in CD56bright and CD56dim NK cells

Soluble HLA-G (sHLA-G) inhibits natural killer (NK) cell functions. Here, we investigated sHLA-G-mediated modulation of (1) chemokine receptor and NK receptor expression and function and (2) cytokine and chemokine secretion in CD56bright and CD56dim NK cells. sHLA-G-treated or untreated peripheral blood (PB) and tonsil NK cells were analyzed for chemokine receptor and NK receptor expression by flow cytometry. sHLA-G down-modulated (1) CXCR3 on PB and tonsil CD56bright and CD56dim, (2) CCR2 on PB and tonsil CD56bright, (3) CX3CR1 on PB CD56dim, (4) CXCR5 on tonsil CD56dim, and (5) CD94/NKG2A on PB and tonsil CD56brigh) and CD56dim NK cells. Such sHLA-G-mediated down-modulations were reverted by adding anti-HLA-G or anti-ILT2 mAbs. sHLA-G inhibited chemotaxis of (1) PB NK cells toward CXCL10, CXCL11, and CX3CL1 and (2) PB CD56bright NK cells toward CCL2 and CXCL10. IFN-γ secretion induced by NKp46 engagement was inhibited by NKG2A engagement in untreated but not in sHLA-G-treated NK cells. sHLA-G up-regulated secretion of (1) CCL22 in CD56bright and CD56dim and (2) CCL2, CCL8, and CXCL2-CXCL3 in CD56dim PB NK cells. Signal transduction experiments showed sHLA-G-mediated down-modulation of Stat5 phosphorylation in PB NK cells. In conclusion, our data delineated novel mechanisms of sHLA-G-mediated inhibition of NK-cell functions.

A novel role of the CX 3 CR1/CX 3 CL1 system in the cross-talk between chronic lymphocytic leukemia cells and tumor microenvironment

Several chemokines/chemokine receptors such as CCR7, CXCR4 and CXCR5 attract chronic lymphocytic leukemia (CLL) cells to specific microenvironments. Here we have investigated whether the CX3CR1/CX3CL1 axis is involved in the interaction of CLL with their microenvironment. CLL cells from 52 patients expressed surface CX3CR1 and CX3CL1 and released constitutively soluble CX3CL1. One third of these were attracted in vitro by soluble CX3CL1. CX3CL1-induced phosphorylation of PI3K, Erk1/2, p38, Akt and Src was involved in induction of CLL chemotaxis. Leukemic B cells upregulated CXCR4 upon incubation with CX3CL1 and this was paralleled by increased chemotaxis to CXCL12. Akt phosphorylation was involved in CX3CL1-induced upregulation of CXCR4 on CLL. In proliferation centers from CLL lymph node and bone marrow, CX3CL1 was expressed by CLL cells whereas CX3CR1 was detected in CLL and stromal cells. Nurselike cells (NLCs) generated from CLL patient blood co-expressed surface CX3CR1 and CX3CL1, but did not secrete soluble CX3CL1. Only half of NLC cell fractions were attracted in vitro by CX3CL1. In conclusion, the CX3CR1/CX3CL1 system may contribute to interactions between CLL cells and tumor microenvironment by increasing CXCL12-mediated attraction of leukemic cells to NLC and promoting directly adhesion of CLL cells to NLC.

Role of Fractalkine/CX3CL1 and Its Receptor in the Pathogenesis of Inflammatory and Malignant Diseases with Emphasis on B Cell Malignancies

Fractalkine/CX3CL1, the only member of the CX3C chemokine family, exists as a membrane-anchored molecule as well as in soluble form, each mediating different biological activities. It is constitutively expressed in many hematopoietic and nonhematopoietic tissues such as endothelial and epithelial cells, lymphocytes, neurons, microglial osteoblasts. The biological activities of CX3CL1 are mediated by CX3CR1, that is expressed on different cell types such as NK cells, CD14+ monocytes, cytotoxic effector T cells, B cells, neurons, microglia, smooth muscle cells, and tumor cells. The CX3CL1/CX3CR1 axis is involved in the pathogenesis of several inflammatory cancer including various B cell malignancies. In tumors the interaction between cancer cells and cellular microenvironment creates a context that may promote tumor growth, increase tumor survival, and facilitate metastasis. Therefore the role of the CX3CL1/CX3CR1 has attracted interest as to the development of potential therapeutic approaches. Here we review the different effects of the CX3CL1/CX3CR1 axis in several inflammatory and neurodegenerative diseases and in cancer, with emphasis on human B cell lymphomas.

Direct inhibition of human acute myeloid leukemia cell growth by IL-12

Acute myeloid leukemia is a haematopoietic malignancy originating from the transformation of myeloid progenitors that proliferate and accumulate in the bone marrow. In AML patients the survival rate at 5 years is 40-50% highlighting the need for novel therapies. In this study we have asked whether IL-12, an immuno-modulatory cytokine with anti-tumor activity, may inhibit directly AML cell growth. We show that the human AML cell lines U937, K562 and THP-1 expressed both chains of the IL-12 receptor (R), i.e. IL-12Rβ1 and IL-12Rβ2. IL-12 inhibited the angiogenic potential of AML cells in vitro, but did not affect their survival or proliferation. In vivo experiments were performed using SCID-NOD mice injected intraperitoneally (i.p.) with the human U937 AML cell line and subsequently treated with human recombinant IL-12 or PBS i.p. Histological, immunohistochemical and flow cytometric analyses on explanted tumors revealed that IL-12 reduced new vessel formation, induced apoptosis and inhibited tumor cell proliferation. Studies on a panel of angiogenesis related genes in explanted tumors using PCR arrays showed significantly down-regulated expression of numerous pro-angiogenic genes including VEGF-C, IL-6, IL-8, CXCL1, CXCL6 and alanyl aminopeptidase in IL-12 vs PBS treated mice. This study shows for the first time that IL-12 targets directly AML cell growth and paves the way to further investigation of IL-12 as potential drug for AML treatment.

CX3CR1 Is Expressed by Human B Lymphocytes and Meditates CX3CL1 Driven Chemotaxis of Tonsil Centrocytes

Background Fractalkine/CX3CL1, a surface chemokine, binds to CX3CR1 expressed by different lymphocyte subsets. Since CX3CL1 has been detected in the germinal centres of secondary lymphoid tissue, in this study we have investigated CX3CR1 expression and function in human naïve, germinal centre and memory B cells isolated from tonsil or peripheral blood. Methodology/Principal Findings We demonstrate unambiguously that highly purified human B cells from tonsil and peripheral blood expressed CX3CR1 at mRNA and protein levels as assessed by quantitative PCR, flow cytometry and competition binding assays. In particular, naïve, germinal centre and memory B cells expressed CX3CR1 but only germinal centre B cells were attracted by soluble CX3CL1 in a transwell assay. CX3CL1 signalling in germinal centre B cells involved PI3K, Erk1/2, p38, and Src phosphorylation, as assessed by Western blot experiments. CX3CR1+ germinal centre B cells were devoid of centroblasts and enriched for centrocytes that migrated to soluble CX3CL1. ELISA assay showed that soluble CX3CL1 was secreted constitutively by follicular dendritic cells and T follicular helper cells, two cell populations homing in the germinal centre light zone as centrocytes. At variance with that observed in humans, soluble CX3CL1 did not attract spleen B cells from wild type mice. OVA immunized CX3CR1−/− or CX3CL1−/− mice showed significantly decreased specific IgG production compared to wild type mice. Conclusion/Significance We propose a model whereby human follicular dendritic cells and T follicular helper cells release in the light zone of germinal centre soluble CX3CL1 that attracts centrocytes. The functional implications of these results warrant further investigation.

Changes in cytokine profile pre- and post-immunosuppression in acquired aplastic anemia

Some patients with aplastic anemia respond to immunosuppressive therapy. Here the authors compare pre and post-immunosuppressive therapy levels of CD3/interefron-γ secreting cells in responders and non-responders. Cytokine expression assessed by flow cytometry in 53 acquired aplastic anemia patients before and after combined immunosuppression (EBMT WPSAA protocols) showed that CD3+ marrow cells containing TNF-α, IFN-γ and IL4 were similar in subjects with disease at onset (DO) and responsive to treatment who had more CD3+/TNF-α+ and CD 3+/IFN-γ+ cells than normal controls. In vitro block of TNF-α and/or IFN-γ significantly increased BFU-e over baseline in 28 patients. In responsive to treatment patients only TNF-α block significantly incremented colonies over normal controls. Absolute marrow CD3+/TNF-α+ and CD3+/IFN-γ+ cells prospectively tested in a group of 21 subjects declined significantly more in Responders than in Non Responders to immunosuppression at Response Evaluation Time respect to Diagnosis. Both in Responders and in Non Responders these cells remained higher than in normal controls. This study suggests that immunosuppression does not fully clear excess TNF-α and IFN-γ from marrow of patients with good outcome and raises the hypothesis that additional cytokine blockade might be useful in immunosuppression for acquired aplastic anemia.

The IL‐31/IL‐31 receptor axis: general features and role in tumor microenvironment

IL‐31 is a recently identified cytokine with a well‐defined role in the pathogenesis of pruritus. IL‐31, whose production is induced by IL‐4 and IL‐33, binds a heterodimeric receptor (R) composed of the exclusive IL‐31RA chain and the shared oncostatin M R. Signaling through the IL‐31R involves the MAPK, PI3K/AKT and Jak/STAT pathways. Different variants and isoforms of IL‐31RA with different signaling activities have been identified. IL‐31 is produced predominantly by circulating Th2 lymphocytes and skin‐homing CLA+CD45RO+ T cells. Studies in humans have demonstrated a pathogenic role for IL‐31 in atopic dermatitis and allergic asthma. The first demonstration of the involvement of the IL‐31/IL‐31R axis in cancer came from studies in patients with mycosis fungoides/Sézary syndrome, the most frequent, cutaneous T cell lymphoma. Tumor cells were shown to produce IL‐31, whose serum levels correlated with pruritus intensity. Follicular lymphoma (FL) B cells and their counterparts—germinal center B cells—produced IL‐31 and expressed IL‐31R, which signaled in the former, but not the latter, cells. IL‐31 released in association with microvesicles promoted tumor growth through autocrine/paracrine loops. Malignant mast cells from patients with mastocytosis or Philadelphia‐negative myeloproliferative disorder produced IL‐31, which contributed to pruritus pathogenesis. Finally, patients with endometrial carcinoma displayed high serum levels of IL‐31 and IL‐33, which may represent promising disease biomarkers. Targeting strategies for the IL‐31/IL‐31R axis have been developed, including the CIMM331 humanized anti‐human IL‐31RA antibody recently tested in a phase I/Ib study.

CX3CL1/fractalkine is a novel regulator of normal and malignant human B cell function

CX3CL1, or fractalkine, the unique member of the CX3C chemokine family, exists as a transmembrane glycoprotein, as well as in soluble form, each mediating different biological activities, and is constitutively expressed in many hematopoietic and nonhematopoietic tissues. CX3CR1, the CX3CL1 exclusive receptor, is a classical GPCR, expressed on NK cells, CD14+ monocytes, and some subpopulation of T cells, B cells, and mast cells. A recent paper by our group has demonstrated for the first time that highly purified human B cells from tonsil and peripheral blood expressed CX3CR1 at mRNA and protein levels. In particular, tonsil naïve, GC, and memory B cells expressed CX3CR1, but only GC centrocytes were attracted by soluble CX3CL1, which with its receptor, are also involved in the pathogenesis of several inflammatory disorders, as well as of cancer. Previous studies have shown that CX3CR1 is up‐regulated in different types of B cell lymphoma, as well as in B‐CLL. Recently, we have demonstrated that the CX3CL1/CX3CR1 axis is involved in the interaction of B‐CLL cells with their microenvironment. Taken together, our data delineate a novel role for the CX3CL1/CX3CR1 complex in the biology of normal B cells and B‐CLL cells. These topics are the subject of this review article.