Boosting the MHC class II-restricted tumor antigen presentation to CD4+ T helper cells: A critical issue for triggering protective immunity and re-orienting the tumor microenvironment toward an anti-tumor state

6noopenAlthough the existence of an immune response against tumor cells is well documented, the fact that tumors take off in cancer patients indicates that neoplastic cells can circumvent this response. Over the years many investigators have described strategies to rescue the anti-tumor immune response with the aim of creating specific and long-lasting protection against the disease. When exported to human clinical settings, these strategies have revealed in most cases a very limited, if any, positive outcome. We believe that the failure is mostly due to the inadequate triggering of the CD4+ T helper (TH) cell arm of the adaptive immunity, as TH cells are necessary to trigger all the immune effector mechanisms required to eliminate tumor cells. In this review, we focus on novel strategies that by stimulating MHC class II-restricted activation of TH cells generate a specific and persistent adaptive immunity against the tumor. This point is of critical importance for both preventive and therapeutic anti-tumor vaccination protocols, because adaptive immunity with its capacity to produce specific, long-lasting protection and memory responses is indeed the final goal of vaccination. We will discuss data from our as well as other laboratories which strongly suggest that triggering a specific and persistent anti-tumor CD4+ TH cell response stably modify not only the tumor microenvironment but also tumor-dependent extratumor microenvironments by eliminating and/or reducing the blood-derived tumor infiltrating cells that may have a pro-tumor growth function such as regulatory CD4+/CD25+ T cells and myeloid-derived-suppressor cells. Within this frame, therefore, we believe that the establishment of a pro-tumor environment is not the cause but simply the consequence of the tumor strategy to primarily counteract components of the adaptive cellular immunity, particularly TH lymphocytes.openAccolla, R.S.; Lombardo, L.; Abdallah, R.; Raval, G.; Forlani, G.; Tosi, G.Accolla, Roberto; Lombardo, L.; Abdallah, R.; Raval, G.; Forlani, Greta; Tosi, Giovann

The human T cell lymphotropic virus 1 (HTLV-1): cellular and molecular characterization of the HTLV-1 oncogenic protein HBZ.

T cell leukemia virus type 1 (HTLV-1 ) is the etiological agent responsible of a severe form of hematologic malignancy designated Adult T cell Leukemia/Lymphoma or ATLL and of aneurological syndrome designated HTLV Associated Myelopathy/Tropical SpasticParaparesis or HAM/TSP. Although the major documented viral oncogenic product ofHTLV-1 is the Tax-1 protein, it has been recently demonstrated that also HBZ (HTLV-1 bZIPfactor), a protein encoded by the minus strand of HTLV-1 genome, is involved in thepathogenesis of ATL. The full role played by HBZ in oncogenesis is still to be explored indetail mainly owing to the unavailability of tools to study this protein in naturally infectedcells and in ATLL cells and its interaction with other crucial cellular proteins involved in thehomeostasis of cell activation and proliferation. By the use of the first reported monoclonalantibody against HBZ generated in our laboratory we have carefully analyzed HBZ proteinexpression, sub-cellular localization as well as its interaction in vivo with endogenous cellfactors in various HBZ-expressing cells, including in particular HTLV-1-infected cells andATLL tumor cell lines. We also demonstrated the ability of this monoclonal antibody to detectHBZ in fresh PBMCs of HTLV-1 infected patients. The availability of this newly generated anti-HBZ mAb has allowed for the first time the quantization of HBZ at the single cell level in naturally infected cells and in neoplastic cells, a better definition of HBZ expression, localization and functional involvement in the biology of HTLV-1 infected cells. The findings described in this thesis may help to better understand the mechanisms through which viral oncogenes contribute to immortalization and neoplasti

The human T cell lymphotropic virus 1 (HTLV-1): cellular and molecular characterization of the HTLV-1 oncogenic protein HBZ.

T cell leukemia virus type 1 (HTLV-1 ) is the etiological agent responsible of a severe form of hematologic malignancy designated Adult T cell Leukemia/Lymphoma or ATLL and of aneurological syndrome designated HTLV Associated Myelopathy/Tropical SpasticParaparesis or HAM/TSP. Although the major documented viral oncogenic product ofHTLV-1 is the Tax-1 protein, it has been recently demonstrated that also HBZ (HTLV-1 bZIPfactor), a protein encoded by the minus strand of HTLV-1 genome, is involved in thepathogenesis of ATL. The full role played by HBZ in oncogenesis is still to be explored indetail mainly owing to the unavailability of tools to study this protein in naturally infectedcells and in ATLL cells and its interaction with other crucial cellular proteins involved in thehomeostasis of cell activation and proliferation. By the use of the first reported monoclonalantibody against HBZ generated in our laboratory we have carefully analyzed HBZ proteinexpression, sub-cellular localization as well as its interaction in vivo with endogenous cellfactors in various HBZ-expressing cells, including in particular HTLV-1-infected cells andATLL tumor cell lines. We also demonstrated the ability of this monoclonal antibody to detectHBZ in fresh PBMCs of HTLV-1 infected patients. The availability of this newly generated anti-HBZ mAb has allowed for the first time the quantization of HBZ at the single cell level in naturally infected cells and in neoplastic cells, a better definition of HBZ expression, localization and functional involvement in the biology of HTLV-1 infected cells. The findings described in this thesis may help to better understand the mechanisms through which viral oncogenes contribute to immortalization and neoplasti

Localization, quantization and interaction with host factors of endogenous HTLV-1 HBZ protein in infected cells and ATL

6sinoneHuman T cell Lymphotropic Virus type 1 (HTLV-1) is the etiological agent of a severe form of neoplasia designated Adult T cell Leukemia/Lymphoma (ATLL) It is widely accepted that the viral transactivator Tax-1 is the major viral product involved in the onset but not in the maintenance of neoplastic phenotype as only 30-40% of ATLL cells express Tax-1. It has been recently demonstrated that HBZ (HTLV-1 bZIP factor), a protein encoded by the minus strand of HTLV-1 genome, constantly expressed in infected cells and in ATLL tumor cells, is also involved in the pathogenesis of leukemia. The full role played by HBZ in oncogenesis is still to be explored in detail mainly owing to the unavailability of tools to assess quantitative expression, subcellular location and interaction of HBZ with host factors in ATLL. By the use of the first reported monoclonal antibody against HBZ, 4D4-F3, generated in our laboratory it has been possible to carefully assess for the first time the above parameters in HTLV-1 chronically infected cells and, most importantly, in leukemic cells from patients. Endogenous HBZ is expressed in speckle-like structures localized in the nucleus. The calculated number of endogenous HBZ molecules varies between18.0 to 36.0 molecules per cell, 22-44 fold less than the amount expressed in HBZ transfected cells used by most investigators to assess the expression, function and subcellular localization of the viral protein. HBZ interacts in vivo with p3 and JunD and co-localizes only partially, and depending on the amount of expressed HBZ, not only with p3 and JunD but also with CBP and CREB2. The possibility to study endogenous HBZ in detail may significantly contribute to a better delineation of the role and presence of HBZ during HTLV-1 infection and cellular transformation.Raval, G.U.; Bidoia, C.; Forlani, G.; Tosi, G.; Gessain, A.; Accolla, R.S.Raval, G. U.; Bidoia, C.; Forlani, Greta; Tosi, Giovanna; Gessain, A.; Accolla, Robert

Cytoplasmic Localization of HTLV-1 HBZ Protein: A Biomarker of HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP)

HTLV-1 is the causative agent of a severe form of adult T cell leukemia/Lymphoma (ATL), and of a chronic progressive neuromyelopathy designated HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). Two important HTLV-1-encoded proteins, Tax-1 and HBZ, play crucial roles in the generation and maintenance of the oncogenic process. Less information is instead available on the molecular and cellular mechanisms leading to HAM/TSP. More importantly, no single specific biomarker has been described that unambiguously define the status of HAM/TSP. Here we report for the first time the finding that HBZ, described until now as an exclusive nuclear protein both in chronically infected and in ATL cells, is instead exclusively localized in the cytoplasm of peripheral blood mononuclear cells (PBMC) from patients suffering of HAM/TSP. Interestingly, at the single cell level, HBZ and Tax-1 proteins are never found co-expressed in the same cell, suggesting the existence of mechanisms of expression uncoupling of these two important HTLV-1 viral products in HAM/TSP patients. Cells expressing cytoplasmic HBZ were almost exclusively found in the CD4+ T cell compartment that was not, at least in a representative HAM/TSP patient, expressing the CD25 marker. Less than 1 percent CD8+ T cells were fond positive for HBZ, while B cells and NK cells were found negative for HBZ in HAM/TSP patients. Our results identify the cytoplasmic localization of HBZ in HAM/TSP patient as a possible biomarker of this rather neglected tropical disease, and raise important hypotheses on the role of HBZ in the pathogenesis of the neuromyelopathy associated to HTLV-1 infection

Das kortikale Netzwerk des auditorischen lexikalischen Zugriffs bei älteren gesunden Erwachsenen

Figure S2. Cell surface phenotype of C5MJ, ATL-2s and patient PH961 cells. Cell surface phenotype of C5MJ, ATL-2s cell lines and of peripheral blood mononuclear cells from ATL patient PH961 was assessed by immunofluorescence and flow cytometry. The various cell surface markers listed in the top right of each histogram were assessed by specific monoclonal antibodies either unlabeled (HLA class I and HLA class II DR) followed by incubation with FITC-labeled rabbit anti-mouse IgG, or directly labeled with fluorochromes (CD3, CD4, CD8, CD25, CD19, and CD16). Specific fluorescence is represented by the bold histogram; negative isotype control is represented by the thin histogram. Values are expressed in the abscissa as mean fluorescence in arbitrary units (au)

Molecular genetics of BCR-ABL1 negative myeloproliferative neoplasms in India

Introduction: Over the past decade, we have moved on from a predominantly morphological and clinical classification of myeloproliferative neoplasms (MPN) to a more evolved classification that accounts for the molecular heterogeneity that is unique to this subgroup of hematological malignancies. This usually incorporates mutations in Janus kinase 2 (JAK2), MPL, and calreticulin (CALR) genes. In this manuscript, we report the frequency of these mutations in a cohort of Indian patients at a tertiary cancer center. Materials and Methods: One hundred and thirty cases of MPN were included in this study. These cases were diagnosed and classified based on the World Health Organization 2008 criteria. JAK2 and MPL mutations were detected using high sensitivity allele-specific polymerase chain reaction using fluorescent labeled primers followed by capillary electrophoresis. A subset of JAK2 and CALR mutations were assessed using a fragment length assay. Results: Among the MPN, we had 20 cases of polycythemia vera (PV), 34 cases of essential thrombocythemia (ET), and 59 of myelofibrosis (MF). JAK2, MPL, and CALR mutations were mutually exclusive of each other. Seventeen cases were categorized as MPN unclassifiable (MPN-U). JAK2p.V617F and MPL mutations were present in 60% (78 of 130) and 5.3% (7 of 130) of all MPN. All the PV cases harbored the JAK2 p.V617F mutation. A total of 23.8% (31 of 130) of patients harbored CALR mutations. CALR exon 9 mutations were detected in 60.8% (14 of 23) and 50% (5 of 10) of JAK2 and MPL negative MF and ET cases, respectively. MPN-U cases included three JAK2 p.V617F positive, two MPL p.W515 L, and 12 CALR positive cases. Ten different types of CALR indels (8 deletions and 2 insertions) were detected of which Type I and Type II mutations were the most common, occurring at a frequency of 45.1% (14 of 31) and 22.5% (7 of 31), respectively. Discussion and Conclusion: We report frequencies of JAK2 p. V617F, MPL exon 10 and CALR mutations in 130 patients similar to those reported in western literature. These mutations carry not only diagnostic but also prognostic relevance

Subcellular localization of endogenous HBZ and Tax-1 in PBMC of HAM/TSP patients.

<p>(A) PBMC of four HAM/TSP patients (PH1485, PH1593, PH1601, and PH1624) were stained with the 4D4-F3 anti-HBZ mAb followed by Alexa Fluor 546-conjugated goat anti-mouse IgG1 antibody (red) and (B) with the A51-2 anti-Tax-1 mAb followed by Alexa Fluor 488-conjugated goat-anti-mouse IgG2a antibody (green), and analyzed by confocal microscopy. Specific counterstaining of nucleus or cytoplasmic compartments was performed by using DRAQ5 fluorescence probe to detect the nucleus and anti-vimentin rabbit polyclonal antibody followed by goat anti-rabbit IgG conjugated to Alexa Fluor 488 (green, panel A) or to Alexa Fluor 546 (red, panel B) to stain the cytoplasmic compartment. At least 300 cells were analyzed; a representative cell for HBZ or for Tax-1 staining is shown for each patient.(C) Low magnification field to show the co-existence of cells mutually exclusive for the expression of cytoplasmic HBZ and Tax-1 in PBMC of patient PH1624. Cells were co-stained with 4D4-F3 anti-HBZ mAb and with A51-2 anti-Tax-1 mAb followed by specific secondary antibodies staining as described in (A) and (B).</p

Subcellular localization of endogenous HBZ and Tax-1 in PBMC of ATL patients.

<p>PBMC of two ATL patients (PH1393 and PH1505) were stained with the anti-HBZ 4D4-F3 mAb, followed by Alexa Fluor 546-conjugated goat anti-mouse IgG1antibody to detect the HBZ protein and analyzed by confocal microscopy. Specific counterstaining of nucleus or cytoplasmic compartments was performed by using DRAQ5 fluorescence probe or anti-vimentin antibody as describe in the legend to <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005285#pntd.0005285.g001" target="_blank">Fig 1</a>. At least 300 cells were analyzed; a representative cell for HBZ staining is shown for each patient. PBMC from both patients were negative for expression of Tax-1 protein.</p

Lack of CD25+ cell in PBMC of patient PH1624, as assessed by confocal microscopy.

<p>PBMC of HAM/TSP patient PH1624 and of healthy control were co-stained with the anti-HBZ 4D4-F3 mAb followed by Alexa Fluor 546-conjugated goat anti-mouse IgG1 antibody (red) and with the anti-CD25 monoclonal antibody directly conjugated to Alexa Fluor 488 (green) and analyzed by confocal microscopy. Upper left panel shows the negativity of CD25 staining in PH1624 patient as compared with a positive cell for the same marker in PBMC of healthy control (PBMC, lower right panel)</p