Stefania Stella

Vδ1 T Lymphocytes from B-CLL Patients Recognize ULBP3 Expressed on Leukemic B Cells and Up-Regulated by Trans-Retinoic Acid

We analyzed 38 untreated patients with chronic lymphocytic leukemia of B-cell type (B-CLL): 24 low-, 8 intermediate-, and 6 high-risk stage. In 15 patients (13 low risk and 2 intermediate risk), circulating Vδ1 T lymphocytes were significantly increased (100 to 300 cells/μL) compared with most intermediate, all high-risk stage, and 15 healthy donors (50 to 100 cells/μL). We studied these Vδ1 T lymphocytes and observed that they proliferated in vitro and produced tumor necrosis factor α or IFN-γ in response to autologous leukemic B cells but not to normal lymphocytes. However, they were unable to kill resting autologous B cells, which lack the MHC-related MIC-A antigen and express low levels of the UL16-binding protein (ULBP) 3 and undetectable levels of ULBP1, ULBP2, and ULBP4. All these molecules are reported ligands for the NKG2D receptor, which is expressed by γδ T cells and activates their cytolytic function. The Vδ1 T lymphocytes studied were able to lyse the ULBP3+ C1R B-cell line upon transfection with MIC-A. More importantly, they also lysed autologous B-CLL cells when transcription and expression of MIC-A or up-regulation of ULBP3 were achieved either by activation or by exposure to trans-retinoic acid. The NKG2D receptor expressed on Vδ1 T cells was involved in the recognition of B-CLL. Finally, in six patients with low numbers of circulating Vδ1 T cells and undetectable ULBP3, the disease progressed over 1 year, whereas no progression occurred in patients with high Vδ1 T lymphocytes and detectable/inducible ULBP3. These data suggest that Vδ1 T lymphocytes may play a role in limiting the progression of B-CLL.

Age-dependent accumulation of monoclonal CD4 + CD8 + double positive T lymphocytes in the peripheral blood of the elderly

Multicolour flow cytometric analysis enabled the identification of monoclonal B‐cell lymphocytosis (MBL), frequently resembling chronic lymphocytic leukaemia, at a rather high frequency in peripheral blood (PB) samples from an elderly population. PB T lymphocytes from 103 otherwise healthy subjects >65 years of age and 51 younger donors (<65 years) were analysed. Besides CD4+ and CD8+ single positive (SP) cells, CD4+CD8+ double positive (DP) mature T lymphocytes were present in both series and could be further distinguished into CD4highCD8low and CD4lowCD8high subsets. An age‐dependent increase of both DP T‐cell subsets was observed, while SP T cells remained stable throughout life. Flow cytometry and polymerase chain reaction analysis of the TRBV expression profiles showed the presence of a TRBV restriction within CD4+CD8+ DP cells in more than half (53/103; 55·3%) of the individuals >65 years of age, regardless the actual number of DP T cells observed. Clonal expansions were more prominent within the CD4highCD8low subset, accounting for most circulating DP clones (47/103; 45·6%). A few cases showed more than one (up to three) monoclonal expansion. Clonal CD4lowCD8high DP T‐lymphocyte expansions were detected in only 10/103 samples (9·7%) and showed a close phenotypic similarity to the rare T‐cell large granular lymphocyte leukaemias. The similarities between DP clones and MBL in the elderly may help to better understand the mechanisms of immunosenescence and their relationships with the development of lymphoproliferative disorders.

The Insulin/IGF System in Colorectal Cancer Development and Resistance to Therapy

The insulin/insulin-like growth factor (IGF) system is a major determinant in the pathogenesis and progression of colorectal cancer (CRC). Indeed, several components of this signaling network, including insulin, IGF-1, IGF-2, the IGF-binding proteins, the insulin receptor (IR), the IGF-1 receptor (IGF-1R), and IR substrate proteins 1 and 2 contribute to the transformation of normal colon epithelial cells. Moreover, the insulin/IGF system is also implicated in the development of resistance to both chemotherapeutic drugs and epidermal growth factor receptor targeted agents. The identification of hybrid receptors comprising both the IR and IGF-1R adds further complexity to this signaling network. Thus, a comprehensive understanding of the biological functions performed by each component of the insulin/IGF system is required to design successful drugs for the treatment of CRC patients.

Influence of complex variant chromosomal translocations in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors

Abstract Cytogenetic variants of the Philadelphia (Ph) chromosome can be observed in 5–8% of patients diagnosed with Chronic Myelogenous Leukemia (CML), and usually involve at least one chromosome other than 9 and 22. Despite the genetically heterogeneous nature of these alterations, available data indicate that CML patients displaying complex variant translocations (CVTs) do not exhibit a less favorable outcome as compared to individuals presenting conventional Ph-positive CML. Patients and methods. We report our experience with 10 CML patients carrying CVTs among 153 newly diagnosed cases followed at our Institution. Results and discussion. Unlike previously published reports, in our series only two CML patients exhibiting CVTs achieved an optimal response to tyrosine kinase inhibitors (TKI) treatment. The remaining eight patients obtained either a suboptimal response or failed drug therapy. Our data suggest that the presence of CVTs at diagnosis might confer an unfavorable clinical outcome, as these genetic alterations might be markers of genomic instability and indicate a higher likelihood of disease progression.

T Cell Receptor (TCR) Gene Transfer with Lentiviral Vectors Allows Efficient Redirection of Tumor Specificity in Naive and Memory T Cells Without Prior Stimulation of Endogenous TCR

We investigated the possibility of introducing exogenous T cell receptor (TCR) genes into T cells by lentiviral transduction, without prior stimulation of endogenous TCR with anti-CD3. TCR transfer is used to impose tumor antigen specificity on recipient T cells, but sustained activation required for retroviral transduction may affect the clinical efficacy of engineered T cells. Cytokine stimulation makes T cells susceptible to lentiviral transduction in the absence of TCR triggering, but this advantage has never been exploited for TCR transfer. Autoimmune diseases are a source of high-affinity TCRs specific for self/tumor antigens. We selected, from a patient with vitiligo, a Mart1-specific TCR based on intrinsic interchain pairing properties and functional avidity. After lentiviral transduction of human peripheral blood mononuclear cells, preferential pairing of exogenous alpha and beta chains was observed, together with effective recognition of Mart1(+) melanoma cells. We tested transduction efficiency on various T cell subsets prestimulated with interleukin (IL)-2, IL-7, IL-15, and IL-21 (alone or in combination). Both naive and unfractionated CD8(+) T cells could be transduced without requiring endogenous TCR triggering. IL-7 plus IL-15 was the most powerful combination, allowing high levels of transgene expression without inducing T cell differentiation (34 +/- 5% Mart1-TCR(+) cells in naive CD8(+) and 16 +/- 6% in unfractionated CD8(+)). Cytokine-prestimulated, Mart1-redirected naive and unfractionated CD8(+) cells expanded better than CD3-CD28-prestimulated counterparts in response to both peptide-pulsed antigen-presenting cells and Mart1(+) melanoma cells. This strategy allows the generation of tumor-specific T cells encompassing truly naive T cells, endowed with an intact proliferative potential and a preserved differentiation stage.

ZAP-70 expression is associated with increased risk of autoimmune cytopenias in CLL patients.

Autoimmune cytopenias (AIC) are frequent in chronic lymphocytic leukemia (CLL) patients, but risk factors and prognostic relevance of these events are controversial. Data about the influence on AIC of biological prognostic markers, as ZAP‐70, are scanty. We retrospectively evaluated AIC in 290 CLL patients tested for ZAP‐70 expression by immunohistochemistry on bone marrow biopsy at presentation. They were 185 men, median age 63 years, 77.9% Binet stage A, 17.6% B and 4.5% C. AIC occurred in 46 patients (16%): 31 autoimmune hemolytic anemias, 10 autoimmune thrombocytopenias, four Evans syndromes, and one pure red cell aplasia. Of the 46 cases of AIC, 37 (80%) occurred in ZAP‐70 positive patients and nine (20%) in ZAP‐70 negatives. ZAP‐70 expression [Hazard Ratio (HR) = 7.42; 95% confidence interval (CI): 2.49–22.05] and age >65 years (HR = 5.41; 95% CI: 1.67–17.49) resulted independent risk factors for AIC. Among the 136 patients evaluated both for ZAP‐70 expression and IGHV status, the occurrence of AIC was higher in ZAP‐70 positive/IGHV unmutated cases (35%) than in patients ZAP‐70 negative/IGHV mutated (6%) or discordant for the two parameters (4%; P < 0.0001). In ZAP‐70 positive patients, occurrence of AIC negatively influenced survival (HR = 1.75; 95% CI: 1.06–2.86). The high risk of developing AIC in ZAP‐70 positive CLL, particularly when IGHV unmutated, should be considered in the clinical management. Am. J. Hematol. 2010.

Suppression of Survivin Induced by a BCR-ABL/JAK2/STAT3 Pathway Sensitizes Imatinib-Resistant CML Cells to Different Cytotoxic Drugs

The BCR-ABL oncoprotein of chronic myelogenous leukemia (CML) displays exclusive cytoplasmic localization and constitutive tyrosine kinase activity leading to the activation of different pathways that favor cell proliferation and survival. BCR-ABL induces survivin expression at both the mRNA and protein level, thus inhibiting the apoptotic machinery of CML cells and contributing to the expansion of the leukemic clone. We report that, in human CML cell lines, BCR-ABL–mediated upregulation of survivin involves the JAK2/STAT3 pathway since silencing of either protein caused a consistent reduction in survivin expression. Cell lines unresponsive to imatinib mesylate (IM) because of BCR-ABL gene amplification were not resensitized to the drug after survivin downregulation. However, cells insensitive to IM because of point mutations in the BCR-ABL kinase domain were highly responsive to hydroxyurea (HU) after survivin silencing. To address the possible clinical applications of our results, we used shepherdin, a cell-permeable peptidomimetic compound that downregulates survivin expression by preventing its interaction with Hsp90. Incubation with shepherdin of immortalized cell lines both sensitive and resistant to IM enhanced cell death induced by HU and doxorubicin. Similarly, the combination of shepherdin with first- and second-generation tyrosine kinase inhibitors reduced the colony-forming potential of human progenitors derived from both patients with IM-sensitive and IM-resistant CML. These results suggest that strategies aimed at reducing survivin levels may represent a potential therapeutic option for patients with CML unresponsive to IM. Mol Cancer Ther; 12(6); 1085–98. ©2013 AACR.

Clonal CD8+ TCR-Vβ expanded populations with effector memory phenotype in Churg Strauss Syndrome

Churg Strauss Syndrome (CSS) is a systemic vasculitis in which oligoclonal T cell expansions might be involved in the pathogenesis. Combined analysis of TCR-Vbeta expression profile by flow cytometry and of TCR gene rearrangement by heteroduplex PCR was used to detect and characterize T cell expansions in 8 CSS patients, 10 asthmatics and 42 healthy subjects. In all CSS patients one or two Vbeta families were expanded among CD8+ cells, with an effector memory phenotype apt to populate tissues and inflammatory sites. Heteroduplex PCR showed the presence of one or more clonal TCR rearrangements, which reveals monoclonal or oligoclonal T cells subpopulations. After purification with a Vbeta specific monoclonal antibody, each CD8+/Vbeta+ expanded family showed a single TCR rearrangement, clearly suggestive of monoclonality. All CD8+ expansions were detectable throughout the disease course. TCR-Vbeta expanded or deleted populations were not observed in asthmatic patients. Clonal CD8+/Vbeta+ T cell expansions might be useful as a disease marker.

Biomarkers and prognostic factors for malignant pleural mesothelioma

The increasing incidence and the dismal prognosis of malignant pleural mesothelioma calls for the identification of biomarkers that will allow a timely diagnosis; display prognostic value; and, predict the response to pharmacological agents employed for the treatment of the disease. Biomarkers associated with early diagnosis currently include mesothelin in combination with miRNA miR-103a-3p. As for prognostic biomarkers, the Cancer and Leukemia Group B (CALGB) and the European Organization for Research and Treatment of Cancer (EORTC) scores take into account different hematological and clinical parameters that distinguish patients with good prognosis from those with inferior outcomes. Fluorodeoxyglucose-PET, microarray expression data, neutrophil-to-lymphocyte ratios, c-MET expression, Ki-67 ratios and fibulin-3 levels have also been associated with disease outcome. Finally, thymidylate synthase protein cutoffs may predict mesothelioma response to the association of pemetrexed with a platinum derivative.

IRF5 is a target of BCR-ABL kinase activity and reduces CML cell proliferation

Interferon regulatory factor 5 (IRF5) modulates the expression of genes controlling cell growth and apoptosis. Previous findings have suggested a lack of IRF5 transcripts in both acute and chronic leukemias. However, to date, IRF5 expression and function have not been investigated in chronic myeloid leukemia (CML). We report that IRF5 is expressed in CML cells, where it interacts with the BCR-ABL kinase that modulates its expression and induces its tyrosine phosphorylation. Tyrosine-phosphorylated IRF5 displayed reduced transcriptional activity that was partially restored by imatinib mesylate (IM). Interestingly, a mutant devoid of a BCR-ABL consensus site (IRF5(Y104F)) still presented significant tyrosine phosphorylation. This finding suggests that the oncoprotein phosphorylates additional tyrosine residues or induces downstream signaling pathways leading to further IRF5 phosphorylation. We also found that ectopic expression of IRF5 decreases the proliferation of CML cell lines by slowing their S-G2 transition, increasing the inhibition of BCR-ABL signaling and enhancing the lethality effect observed after treatment with IM, α-2-interferon and a DNA-damaging agent. Furthermore, IRF5 overexpression successfully reduced the clonogenic ability of CML CD34-positive progenitors before and after exposure to the above-indicated cytotoxic stimuli. Our data identify IRF5 as a downstream target of the BCR-ABL kinase, suggesting that its biological inactivation contributes to leukemic transformation.