Paola Circosta

MEC1 and MEC2: two new cell lines derived from B-chronic lymphocytic leukaemia in prolymphocytoid transformation

We report the establishment and characterization of two cell lines, MEC1 and MEC2, that grew spontaneously on two subsequent occasions from the peripheral blood (PB) of a patient with B-chronic lymphocytic leukemia (B-CLL) in prolymphocytoid transformation. The patient was EBV-seropositive, his leukemic cells were EBNA negative, but the spontaneously grown cell lines are EBNA-2 positive. In liquid culture MEC1 cells grow adherent to the vessel wall and as tiny clumps; MEC2 cells do not adhere and form large clumps. The doubling time of MEC1 is 40h and of MEC2 is 31h. Both cell lines express the same light (kappa) and heavy chains (mu, delta) as the fresh parental B-CLL cells at the same high intensity, share the expression of mature B cell markers (CD19, CD20, CD21, CD22), differ in the expression of CD23 and FMC7, are CD11a+, CD18+, CD44+, CD49d+, CD54+ and express at high levels both CD80 and CD86. CD5 is negative on MEC1 cells (as on the vast majority of parental cells) and it has been lost by MEC2 cells after several months of culture. The cells have a complex karyotype. The tumour origin of MEC1 and MEC2 has been demonstrated by Southern blot analysis of the IgH loci and by Ig gene DNA sequencing. They use the VH4 Ig family and have not undergone somatic mutations (94.8% homology with germline Ig gene 4-59). Cytofluorographic analysis and RT-PCR reveal that MEC1 and MEC2 overexpress Bcl-2 together with Bax, express large amounts of Bcl-xL and trace amounts of Bcl-xS.

Differential Effects on CLL Cell Survival Exerted by Different Microenvironmental Elements

Selected microenvironmental stimuli confer to leukemic cells a growth advantage and an extended survival. We aimed at dissecting the differential support provided by the different cellular components of the microenvironment where CLL cells accumulate. To this end we cultured purified CLL cells in vitro in the presence or absence of different accessory cells (stromal cells, autologous T lymphocytes) and/or soluble molecules (IL-4, sCD40L) and assessed the leukemic cell response in terms of cell viability and chemoattracting capacity. The results indicate that both T lymphocytes and stromal cells are involved in sustaining the survival of leukemic B cells, but indicate that their support is different in terms of time of onset and duration. T cells have a short-term support activity while stromal cells provide long-term support.

Leukemia-Derived Immature Dendritic Cells Differentiate into Functionally Competent Mature Dendritic Cells That Efficiently Stimulate T Cell Responses

Primary acute myeloid leukemia cells can be induced to differentiate into dendritic cells (DC). In the presence of GM-CSF, TNF-α, and/or IL-4, leukemia-derived DC are obtained that display features of immature DC (i-DC). The aim of this study was to determine whether i-DC of leukemic origin could be further differentiated into mature DC (m-DC) and to evaluate the possibility that leukemic m-DC could be effective in vivo as a tumor vaccine. Using CD40L as maturating agent, we show that leukemic i-DC can differentiate into cells that fulfill the phenotypic criteria of m-DC and, compared with normal counterparts, are functionally competent in vitro in terms of: 1) production of cytokines that support T cell activation and proliferation and drive Th1 polarization; 2) generation of autologous CD8+ CTLs and CD4+ T cells that are MHC-restricted and leukemia-specific; 3) migration from tissues to lymph nodes; 4) amplification of Ag presentation by monocyte attraction; 5) attraction of naive/resting and activated T cells. Irradiation of leukemic i-DC after CD40L stimulation did not affect their differentiating and functional capacity. Our data indicate that acute myeloid leukemia cells can fully differentiate into functionally competent m-DC and lay the ground for testing their efficacy as a tumor vaccine.

High Basal γH2AX Levels Sustain Self‐Renewal of Mouse Embryonic and Induced Pluripotent Stem Cells

Phosphorylation of histone H2AX (γH2AX) is known to be the earliest indicator of DNA double‐strand breaks. Recently, it has been shown that mouse embryonic stem cells (mESCs) have very high basal levels of γH2AX, even when they have not been exposed to genotoxic agents. As the specialized role of high basal γH2AX levels in pluripotent stem cells is still debated, we investigated whether H2AX phosphorylation is important in maintaining self‐renewal of these cells. Here, we report that not only mESCs but also mouse‐induced pluripotent stem cells (miPSCs), have high basal levels of γH2AX. We show that basal γH2AX levels decrease upon ESC and iPSC differentiation and increase when the cells are treated with self‐renewal‐enhancing small molecules. We observe that self‐renewal activity is highly compromised in H2AX−/− cells and that it can be restored in these cells through reconstitution with a wild‐type, but not a phospho‐mutated, H2AX construct. Taken together, our findings suggest a novel function of H2AX that expands the knowledge of this histone variant beyond its role in DNA damage and into a new specialized biological function in mouse pluripotent stem cells. STEM CELLS2012;30:1414–1423

miR-223 is a coordinator of breast cancer progression as revealed by bioinformatics predictions.

MicroRNAs are single-stranded non-coding RNAs that simultaneously down-modulate the expression of multiple genes post-transcriptionally by binding to the 3′UTRs of target mRNAs. Here we used computational methods to predict microRNAs relevant in breast cancer progression. Specifically, we applied different microRNA target prediction algorithms to various groups of differentially expressed protein-coding genes obtained from four breast cancer datasets. Six potential candidates were identified, among them miR-223, previously described to be highly expressed in the tumor microenvironment and known to be actively transferred into breast cancer cells. To investigate the function of miR-223 in tumorigenesis and to define its molecular mechanism, we overexpressed miR-223 in breast cancer cells in a transient or stable manner. Alternatively we overexpressed miR-223 in mouse embryonic fibroblasts or HEK293 cells and used their conditioned medium to treat tumor cells. With both approaches, we obtained elevated levels of miR-223 in tumor cells and observed decreased migration, increased cell death in anoikis conditions and augmented sensitivity to chemotherapy but no effect on adhesion and proliferation. The analysis of miR-223 predicted targets revealed enrichment in cell death and survival-related genes and in pathways frequently altered in breast cancer. Among these genes, we showed that protein levels for STAT5A, ITGA3 and NRAS were modulated by miR-223. In addition, we proved that STAT5A is a direct miR-223 target and highlighted a possible correlation between miR-223 and STAT5A in migration and chemotherapy response. Our investigation revealed that a computational analysis of cancer gene expression datasets can be a relevant tool to identify microRNAs involved in cancer progression and that miR-223 has a prominent role in breast malignancy that could potentially be exploited therapeutically.

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.

The characterization of chemokine production and chemokine receptor expression reveals possible functional cross-talks in AML blasts with monocytic differentiation

OBJECTIVE The mechanisms regulating the trafficking of leukemic myeloid blasts are poorly understood. A differential expression of chemokines and chemokine receptors might account for some aspects of the pattern of invasion and accumulation of leukemic cells. We aimed at defining the pattern of chemokine and chemokine receptor expression of acute myeloid leukemia (AML) blasts in comparison with their putative normal cell counterparts. PATIENTS AND METHODS Twenty-five cases of AML were analyzed by flow cytometry for the expression of several chemokine receptors and by RT-PCR for the expression of relevant chemokines. For selected chemokines, the production was confirmed by ELISA. AML blasts were also assessed for their migration capacity in response to autologous supernatants and recombinant chemokines. RESULTS Undifferentiated AML (MO-M1 and some M2) express only CXCR4 on their surface and produce mainly inflammatory chemokines, resembling normal CD34+ progenitors. More differentiated AML (M4-M5 and some M2) have a more diversified receptor repertoire and, besides CXCR4, express the receptors for inflammatory chemokines and produce both constitutive and inflammatory chemokines, resembling resting and activated monocytes. In particular, M4-M5 blasts produce MCP-1 and MIP-3alpha and also express their specific receptors (CCR2 and, to a lesser extent, CCR6) and migrate in vitro in response to MCP-1 and MIP-3alpha and to their own supernatant. A significant correlation between extramedullary involvement and coexpression of MCP-1/CCR2 was found. CONCLUSIONS These data suggest that chemokines and their receptors segregate within the different FAB subtypes and, by allowing cross-talk among members of the malignant clone, might help to explain some aspects of the pattern of invasion in AML with monocytic differentiation.

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.

The molecular and functional characterization of clonally expanded CD8+ TCR BV T cells in eosinophilic granulomatosis with polyangiitis (EGPA)

In eosinophilic granulomatosis with polyangiitis (EGPA) clonally expanded T cells might concur in granuloma formation and vascular injury. The TCR β-variable (BV) chain repertoire and third complementarity determining region (CDR3) of peripheral CD4+ and CD8+ cells in EGPA patients and age-matched controls and the expression of cytokines and chemokine receptors were investigated. The CD8+ lymphocytes of EGPA patients showed an increased frequency of BV expansions with a skewed profile of BV CDR3 lengths, increased CCR5 and CXCR3 expression and increased INFγ and TNFα production. In two patients, the TCR CDR3 cDNA sequences of the expanded BV family were identified. The CD4+ lymphocytes of EGPA patients revealed a higher expression of CRTH2 and increased production of IL-5. In conclusion, CD4+ T cells display a Th2 profile and CD8+ T cells are clonally expanded in EGPA and have a proinflammatory phenotype, suggesting their pathogenic role in vasculitic damage.

Transient proteasome inhibition as a strategy to enhance lentiviral transduction of hematopoietic CD34(+) cells and T lymphocytes: implications for the use of low viral doses and large-size vectors.

The proteasome system restricts lentiviral transduction of stem cells. We exploited proteasome inhibition as a strategy to enhance transduction of both hematopoietic stem cells (HSC) and T lymphocytes with low dose or large-size lentiviral vectors (LV). HSC showed higher transduction efficiency if transiently exposed to proteasome inhibitor MG132 (41.8% vs 10.7%, p<0.0001). Treatment with MG132 (0.5 μM) retained its beneficial effect with 3 different LV of increasing size up to 10.9 Kb (p<0.01). We extended, for the first time, the application of proteasome inhibition to the transduction of T lymphocytes. A transient exposure to MG132 significantly improved lentiviral T-cell transduction. The mean percentage of transduced T cells progressively increased from 13.5% of untreated cells, to 21% (p=0.3), 30% (p=0.03) and 37% (p=0.01) of T lymphocytes that were pre-treated with MG132 at 0.1, 0.5 and 1 μM, respectively. MG132 did not affect viability or functionality of HSC or T cells, nor significantly increased the number of integrated vector copies. Transient proteasome inhibition appears as a new procedure to safely enhance lentiviral transduction of HSC and T lymphocytes with low viral doses. This approach could be useful in settings where the use of large size vectors may impair optimal viral production.