Maria Corallo

Cells with Characteristics of Cancer Stem/Progenitor Cells Express the CD133 Antigen in Human Endometrial Tumors

Purpose: Cancer stem cells represent an attractive therapeutic target for tumor eradication. The present study aimed to determine whether CD133 expression may identify cells with characteristics of cancer stem/progenitor cells in human endometrial tumors. Experimental Design: We analyzed 113 tumor samples for CD133/1 expression by flow cytometry, immunohistochemistry, and semiquantitative reverse transcription–PCR. CD133+ cells were isolated and used to assess phenotypic characteristics, self-renewal capacity, ability to maintain CD133 expression and form sphere-like structures in long-term cultures, sensitivity to chemotherapeutic agents, gene expression profile, and ability to initiate tumors in NOD/SCID mice. Results: Primary tumor samples exhibited a variable degree of immunoreactivity for CD133/1, ranging from 1.3% to 62.6%, but stained negatively for other endothelial and stem cell–associated markers. Isolated CD133+ cells expanded up to 4.6-fold in serum-replenished cultures and coexpressed the GalNAcα1-O-Ser/Thr MUC-1 glycoform, a well-characterized tumor-associated antigen. Dissociated bulk tumors formed sphere-like structures; cells grown as tumor spheres maintained CD133 expression and could be propagated for up to 12 weeks. CD133+ cells purified from endometrioid adenocarcinomas were resistant to cisplatin-induced and paclitaxel-induced cytotoxicity and expressed a peculiar gene signature consisting of high levels of matrix metalloproteases, interleukin-8, CD44, and CXCR4. When serially transplanted into NOD/SCID mice, CD133+ cells were capable of initiating tumor formation and recapitulating the phenotype of the original tumor. Conclusions: CD133 is expressed by human endometrial cancers and might represent a valuable tool to identify cells with cancer stem cell characteristics.

Human cord blood CD133+ cells immunoselected by a clinical-grade apparatus differentiate in vitro into endothelial- and cardiomyocyte-like cells

BACKGROUND: Recent findings on human hematopoietic stem cell (HSC) properties suggest a possible therapeutic role of human umbilical cord blood (UCB) HSC‐based cellular therapies in the treatment of myocardial infarction.

Thymoglobulin, interferon-γ and interleukin-2 efficiently expand cytokine-induced killer (CIK) cells in clinical-grade cultures

BackgroundCytokine-induced killer (CIK) cells are typically differentiated in vitro with interferon (IFN)-γ and αCD3 monoclonal antibodies (mAb), followed by the repeated provision of interleukin (IL)-2. It is presently unknown whether thymoglobulin (TG), a preparation of polyclonal rabbit γ immunoglobulins directed against human thymocytes, can improve the generation efficiency of CIK cells compared with αCD3 mAb in a clinical-grade culture protocol.MethodsPeripheral blood mononuclear cells (PBMC) from 10 healthy donors and 4 patients with solid cancer were primed with IFN-γ on day 0 and low (50 ng/ml), intermediate (250 ng/ml) and high (500 ng/ml) concentrations of either αCD3 mAb or TG on day 1, and were fed with IL-2 every 3 days for 21 days. Aliquots of cells were harvested weekly to monitor the expression of representative members of the killer-like immunoglobulin receptor (KIR), NK inhibitory receptor, NK activating receptor and NK triggering receptor families. We also quantified the frequency of bona fide regulatory T cells (Treg), a T-cell subset implicated in the down-regulation of anti-tumor immunity, and tested the in vitro cytotoxic activity of CIK cells against NK-sensitive, chronic myeloid leukaemia K562 cells.ResultsCIK cells expanded more vigorously in cultures supplemented with intermediate and high concentrations of TG compared with 50 ng/ml αCD3 mAb. TG-driven CIK cells expressed a constellation of NK activating/inhibitory receptors, such as CD158a and CD158b, NKp46, NKG2D and NKG2A/CD94, released high quantities of IL-12p40 and efficiently lysed K562 target cells. Of interest, the frequency of Treg cells was lower at any time-point compared with PBMC cultures nurtured with αCD3 mAb. Cancer patient-derived CIK cells were also expanded after priming with TG, but they expressed lower levels of the NKp46 triggering receptor and NKG2D activating receptor, thus manifesting a reduced ability to lyse K562 cells.ConclusionsTG fosters the generation of functional CIK cells with no concomitant expansion of tumor-suppressive Treg cells. The culture conditions described herein should be applicable to cancer-bearing individuals, although the differentiation of fully functional CIK cells may be hindered in patients with advanced malignancies.

Effects of pegylated G-CSF on immune cell number and function in patients with gynecological malignancies

BackgroundPegylated granulocyte colony-stimulating factor (G-CSF; pegfilgrastim) is a longer-acting form of G-CSF, whose effects on dendritic cell (DC) and regulatory T cell (Treg) mobilization, and on the in vivo and ex vivo release of immune modulating cytokines remain unexplored.MethodsTwelve patients with gynecological cancers received carboplatin/paclitaxel chemotherapy and single-dose pegfilgrastim as prophylaxis of febrile neutropenia. Peripheral blood was collected prior to pegfilgrastim administration (day 0) and on days +7, +11 and +21, to quantify immunoregulatory cytokines and to assess type 1 DC (DC1), type 2 DC (DC2) and Treg cell mobilization. In vitro-differentiated, monocyte-derived DC were used to investigate endocytic activity, expression of DC maturation antigens and ability to activate allogeneic T-cell proliferation.ResultsPegfilgrastim increased the frequency of circulating DC1 and DC2 precursors. In contrast, CD4+FoxP3+bona fide Treg cells were unchanged compared with baseline. Serum levels of hepatocyte growth factor and interleukin (IL)-12p40, but not transforming growth factor-β1 or immune suppressive kynurenines, significantly increased after pegfilgrastim administration. Interestingly, pegfilgrastim fostered in vitro monocytic secretion of IL-12p40 and IL-12p70 when compared with unconjugated G-CSF. Finally, DC populations differentiated in vitro after clinical provision of pegfilgrastim were phenotypically mature, possessed low endocytic activity, and incited a robust T-cell proliferative response.ConclusionsPegfilgrastim induced significant changes in immune cell number and function. The enhancement of monocytic IL-12 secretion portends favorable implications for pegfilgrastim administration to patients with cancer, a clinical context where the induction of immune deviation would be highly undesirable.

Interleukin-21 induces the differentiation of human umbilical cord blood CD34-lineage- cells into pseudomature lytic NK cells

BackgroundUmbilical cord blood (UCB) is enriched with transplantable CD34+ cells. In addition to CD34-expressing haematopoietic stem cells (HSC), human UCB contains a rare population of CD34-lineage- cells endowed with the ability to differentiate along the T/NK pathway in response to interleukin (IL)-15 and a stromal cell support. IL-21 is a crucial regulator of NK cell function, whose influence on IL-15-induced differentiation of CD34-lineage- cells has not been investigated previously. The present study was designed and conducted to address whether IL-21 might replace the stromal cell requirements and foster the IL-15-induced NK differentiation of human UCB CD34-lineage- cells.ResultsCD34-lineage- cells were maintained in liquid culture with Flt3-L and SCF, with the addition of IL-15 and IL-21, either alone or in combination. Cultures were established in the absence of feeder cells or serum supplementation. Cytokine-treated cells were used to evaluate cell surface phenotype, expression of molecular determinants of lymphoid/NK cell differentiation, secretion of IFN-γ, GM-CSF, TNF-α and CCL3/MIP-1α, and cytolytic activity against NK-sensitive tumour cell targets. CD34-lineage- cells proliferated vigorously in response to IL-15 and IL-21 but not to IL-21 alone, and up-regulated phosphorylated Stat1 and Stat3 proteins. CD34-lineage- cells expanded by IL-21 in combination with IL-15 acquired lymphoid morphology and killer-cell immunoglobulin-like receptor (KIR)-CD56+CD16-/+ phenotype, consistent with pseudo-mature NK cells. IL-21/IL-15-differentiated cells expressed high levels of mRNA for Bcl-2, GATA-3 and Id2, a master switch required for NK-cell development, and harboured un-rearranged TCRγ genes. From a functional standpoint, IL-21/IL-15-treated cells secreted copious amounts of IFN-γ, GM-CSF and CCL3/MIP-1α, and expressed cell surface CD107a upon contact with NK-sensitive tumour targets, a measure of exocytosis of NK secretory granules.ConclusionThis study underpins a novel role for IL-21 in the differentiation of pseudo-mature lytic NK cells in a synergistic context with IL-15, and identifies a potential strategy to expand functional NK cells for immunotherapy.

Source of cell injected is a critical factors for short and long engraftment in xeno-transplantation.

Abstract. This study aims to investigate engraftment of human cord blood and foetal bone marrow stem cells after in utero transplantation via the intracoelomic route in the sheep. Here, we performed transplantation in 14 single and 1 twin sheep foetuses at 40–47 days of development, using a novel schedule for injection. (i) Single injection of CD34+ human cord blood stem cells via the coelomic route (from 10 to 50 × 104) in seven single foetuses. (ii) Single injection of CD34+ foetal bone marrow stem cells via the intracoelomic route with further numbers of cells (20 × 105 and 8 × 105, respectively) in three single and in one twin foetuses. (iii) Double fractioned injection (20–30 × 106) via the coelomic route and 20 × 106 postnatally, intravenously, shortly after birth of CD3‐depleted cord blood stem cells in four single foetuses. In the first group, three single foetuses showed human/sheep chimaerism at 1, 8 and 14 months after birth. In the second group, the twin foetuses showed human/sheep chimaerism at 1 month after birth. In the third group, only two out of four single foetuses that underwent transplantation showed chimaerism at 1 month. While foetal bone marrow stem cells showed good short‐term engraftment (1 month after birth), cord blood stem cells were able to persist longer in the ovine recipients (at 1, 8 and 14 months after birth).