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author:("labelle, Anna")
1.  Protein Kinase CK2 Inhibition Down Modulates the NF-κB and STAT3 Survival Pathways, Enhances the Cellular Proteotoxic Stress and Synergistically Boosts the Cytotoxic Effect of Bortezomib on Multiple Myeloma and Mantle Cell Lymphoma Cells 
PLoS ONE  2013;8(9):e75280.
CK2 is a pivotal pro-survival protein kinase in multiple myeloma that may likely impinge on bortezomib-regulated cellular pathways. In the present study, we investigated CK2 expression in multiple myeloma and mantle cell lymphoma, two bortezomib-responsive B cell tumors, as well as its involvement in bortezomib-induced cytotoxicity and signaling cascades potentially mediating bortezomib resistance. In both tumors, CK2 expression correlated with that of its activated targets NF-κB and STAT3 transcription factors. Bortezomib-induced proliferation arrest and apoptosis were significantly amplified by the simultaneous inhibition of CK2 with two inhibitors (CX-4945 and K27) in multiple myeloma and mantle cell lymphoma cell lines, in a model of multiple myeloma bone marrow microenvironment and in cells isolated from patients. CK2 inhibition empowered bortezomib-triggered mitochondrial-dependent cell death. Phosphorylation of NF-κB p65 on Ser529 (a CK2 target site) and rise of the levels of the endoplasmic reticulum stress kinase/endoribonuclease Ire1α were markedly reduced upon CK2 inhibition, as were STAT3 phospho Ser727 levels. On the contrary, CK2 inhibition increased phospho Ser51 eIF2α levels and enhanced the bortezomib-dependent accumulation of poly-ubiquitylated proteins and of the proteotoxic stress-associated chaperone Hsp70. Our data suggest that CK2 over expression in multiple myeloma and mantle cell lymphoma cells might sustain survival signaling cascades and can antagonize bortezomib-induced apoptosis at different levels. CK2 inhibitors could be useful in bortezomib-based combination therapies.
doi:10.1371/journal.pone.0075280
PMCID: PMC3785505  PMID: 24086494
2.  HS1, a Lyn Kinase Substrate, Is Abnormally Expressed in B-Chronic Lymphocytic Leukemia and Correlates with Response to Fludarabine-Based Regimen 
PLoS ONE  2012;7(6):e39902.
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.
doi:10.1371/journal.pone.0039902
PMCID: PMC3387232  PMID: 22768161
3.  Identification of a CD11b+/Gr-1+/CD31+ myeloid progenitor capable of activating or suppressing CD8+ T cells 
Blood  2000;96(12):3838-3846.
Apoptotic death of CD8+ T cells can be induced by a population of inhibitory myeloid cells that are double positive for the CD11b and Gr-1 markers. These cells are responsible for the immunosuppression observed in pathologies as dissimilar as tumor growth and overwhelming infections, or after immunization with viruses. The appearance of a CD11b+/Gr-1+ population of inhibitory macrophages (i Macs) could be attributed to high levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) in vivo. Deletion of i Macs in vitro or in vivo reversed the depression of CD8+ T-cell function. We isolated i Macs from the spleens of immunocompromised mice and found that these cells were positive for CD31, ER-MP20 (Ly-6C), and ER-MP58, markers characteristic of granulocyte/monocyte precursors. Importantly, although i Macs retained their inhibitory properties when cultured in vitro in standard medium, suppressive functions could be modulated by cytokine exposure. Whereas culture with the cytokine interleukin 4 (IL-4) increased i Mac inhibitory activity, these cells could be differentiated into a nonadherent population of fully mature and highly activated dendritic cells when cultured in the presence of IL-4 and GM-CSF. A common CD31+/CD11b+/Gr-1+ progenitor can thus give rise to cells capable of either activating or inhibiting the function of CD8+ T lymphocytes, depending on the cytokine milieu that prevails during antigen-presenting cell maturation.
PMCID: PMC2734459  PMID: 11090068
4.  Unopposed Production of Granulocyte-Macrophage Colony-Stimulating Factor by Tumors Inhibits CD8+ T Cell Responses by Dysregulating Antigen-Presenting Cell Maturation1 
Tumor cells gene-modified to produce GM-CSF potently stimulate antitumor immune responses, in part, by causing the growth and differentiation of dendritic cells (DC). However, GM-CSF-modified tumor cells must be γ-irradiated or they will grow progressively, killing the host. We observed that 23 of 75 (31%) human tumor lines and two commonly used mouse tumor lines spontaneously produced GM-CSF. In mice, chronic GM-CSF production by tumors suppressed Ag-specific CD8+ T cell responses. Interestingly, an inhibitory population of adherent CD11b(Mac-1)/Gr-1 double-positive cells caused the observed impairment of CD8+ T cell function upon direct cell-to-cell contact. The inhibitory cells were positive for some markers associated with Ag presenting cells, like F4/80, but were negative for markers associated with fully mature DC like DEC205, B7.2, and MHC class II. We have previously reported that a similar or identical population of inhibitory “immature” APC was elicited after immunization with powerful recombinant immunogens. We show here that these inhibitory cells can be elicited by the administration of recombinant GM-CSF alone, and, furthermore, that they can be differentiated ex vivo into “mature” APC by the addition of IL-4 and GM-CSF. Thus, tumors may be able to escape from immune detection by producing “unopposed” GM-CSF, thereby disrupting the balance of cytokines needed for the maturation of fully functional DC. Further, CD11b/Gr-1 double-positive cells may function as “inhibitory” APC under the influence of GM-CSF alone.
PMCID: PMC2228333  PMID: 10229805
5.  The neutrophil-activating protein of Helicobacter pylori promotes Th1 immune responses 
Journal of Clinical Investigation  2006;116(4):1092-1101.
The Helicobacter pylori neutrophil-activating protein (HP-NAP) is a virulence factor of H. pylori that stimulates in neutrophils high production of oxygen radicals and adhesion to endothelial cells. We report here that HP-NAP is a TLR2 agonist able to induce the expression of IL-12 and IL-23 by neutrophils and monocytes. Addition in culture of HP-NAP, as an immune modulator, to antigen-induced T cell lines resulted in a remarkable increase in the number of IFN-γ–producing T cells and decrease of IL-4–secreting cells, thus shifting the cytokine profile of antigen-activated human T cells from Th2 to a Th1 cytotoxic phenotype. We also found that in vivo HP-NAP elicited an antigen-specific Th1-polarized T cell response in the gastric mucosa of H. pylori–infected patients. These data indicate HP-NAP as an important factor of H. pylori able to elicit cells of the innate immune system to produce IL-12 and IL-23, and they suggest it as a new tool for promoting Th1 immune responses.
doi:10.1172/JCI27177
PMCID: PMC1401483  PMID: 16543949
6.  CXCR3/CXCL10 interactions in the development of hypersensitivity pneumonitis 
Respiratory Research  2005;6(1):20.
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(+)/IFNγ(+) Tc1 cells exhibiting a strong in vitro migratory capability in response to CXCL10. Alveolar macrophages expressed and secreted, in response to IFN-γ, 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-γ mediates the recruitment of lymphocytes into the lung via production of the chemokine CXCL10, resulting in Tc1-cell alveolitis and granuloma formation.
doi:10.1186/1465-9921-6-20
PMCID: PMC554979  PMID: 15725351
7.  Chronic lymphocytic leukemia B cells contain anomalous Lyn tyrosine kinase, a putative contribution to defective apoptosis 
Journal of Clinical Investigation  2005;115(2):369-378.
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.
doi:10.1172/JCI200522094
PMCID: PMC544036  PMID: 15650771
8.  CXCR3/CXCL10 expression in the synovium of children with juvenile idiopathic arthritis 
Arthritis Research & Therapy  2005;7(2):R241-R249.
The accumulation of T cells in the synovial membrane is the crucial step in the pathophysiology of the inflammatory processes characterizing juvenile idiopathic arthritis (JIA). In this study, we evaluated the expression and the pathogenetic role in oligoarticular JIA of a CXC chemokine involved in the directional migration of activated T cells, i.e. IFNγ-inducible protein 10 (CXCL10) and its receptor, CXCR3. Immunochemistry with an antihuman CXCL10 showed that synovial macrophages, epithelial cells, and endothelial cells bear the chemokine. By flow cytometry and immunochemistry, it has been shown that CXCR3 is expressed at high density by virtually all T lymphocytes isolated from synovial fluid (SF) and infiltrating the synovial membrane. Particularly strongly stained CXCR3+ T cells can be observed close to the luminal space and in the perivascular area. Furthermore, densitometric analysis has revealed that the mRNA levels for CXCR3 are significantly higher in JIA patients than in controls. T cells purified from SF exhibit a definite migratory capability in response to CXCL10. Furthermore, SF exerts significant chemotactic activity on the CXCR3+ T-cell line, and this activity is inhibited by the addition of an anti-CXCL10 neutralizing antibody. Taken together, these data suggest that CXCR3/CXCL10 interactions are involved in the pathophysiology of JIA-associated inflammatory processes, regulating both the activation of T cells and their recruitment into the inflamed synovium.
doi:10.1186/ar1481
PMCID: PMC1065320  PMID: 15743470
chemokines; CXCL10; juvenile idiopathic arthritis; pathogenesis

Results 1-8 (8)