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Supplementary Data from 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.

BACKGROUND Mesenchymal stromal cells (MSCs) isolated from human umbilical cord tissue (UCT) can be considered the perfect candidates for cell-based therapies and regenerative medicine. UCT-derived MSCs can be cryogenically stored in cell banks and expanded as needed for therapeutic uses. STUDY DESIGN AND METHODS We developed a new method for UCT-MSC isolation, cryopreservation, and expansion, following all criteria required by a stem cell bank. UCT-MSCs were isolated either by manual dissociation (MM) or by a semiautomatic dissociation system (SAM). In both protocols UCTs were treated enzymatically using Type IV collagenase good manufacturing practices (GMP) graded and hyaluronidase (medicinal product). Isolated UCT-MSCs were cryopreserved and analyzed after thawing for phenotype; for proliferation rate; and for their osteogenic, adipogenic, and chondrogenic differentiation capabilities. RESULTS We found that SAM reduced the time of tissue enzyme exposure and enabled us to obtain a homogeneous single-cell suspension deprived of tissue fragments. The isolated cells in both groups showed high expression of MSC markers CD105, CD73, and CD90 and similar differentiation capabilities, phenotype, and proliferation potential. Moreover, the final yield of MSCs was comparable between the two techniques. CONCLUSION In this study, we have established a reliable and standardized protocol to isolate UCT-MSCs from UCT for cell banking purposes. Processing the whole umbilical tissue with GMP-graded enzymes using a semiautomatic dissociator allowed us to obtain a single-cell suspension product with a known number of isolated cells that can be cryopreserved right after isolation and thawed as needed for expansion and clinical use.

Alternatives to the use of fetal bovine serum (FBS) have been investigated to ensure xeno-free growth condition. In this study we evaluated the efficacy of human platelet lysate (PL) as a substitute of FBS for the in vitro culture of some human cell lines. PL was obtained by pools of pathogen inactivated human donor platelet (PLT) concentrates. Human leukemia cell lines (KG-1, K562, JURKAT, HL-60) and epithelial tumor cell lines (HeLa and MCF-7) were cultured with either FBS or PL. Changes in cell proliferation, viability, morphology, surface markers and cell cycle were evaluated for each cell line. Functional characteristics were analysed by drug sensitivity test and cytotoxicity assay. Our results demonstrated that PL can support growth and expansion of all cell lines, although the cells cultured in presence of PL experienced a less massive proliferation compared to those grown with FBS. We found a comparable percentage of viable specific marker-expressing cells in both conditions, confirming lineage fidelity in all cultures. Functionality assays showed that cells in both FBS- and PL-supported cultures maintained their normal responsiveness to adriamycin and NK cell-mediated lysis. Our findings indicate that PL is a feasible serum substitute for supporting growth and propagation of haematopoietic and epithelial cell lines with many advantages from a perspective of process standardization, ethicality and product safety.

The adverse biological effects of continuous exposure to cobalt and chromium have been well defined. In the past, this toxicity was largely an industrial issue concerning workers exposed in occupational setting. Nevertheless, recent reports have described a specific toxicity mediated by the high levels of cobalt and chromium released by metallic prostheses, particularly in patients who had received hip implants. Clinical symptoms, including blindness, deafness and peripheral neuropathy, suggest a specific neurotropism. However, little is known about the neuropathological basis of this process, and experimental evidence is still lacking. We have investigated this issue in an experimental setting using New Zealand White rabbits treated with repeated intravenous injections of cobalt and chromium, alone or in combination. No evident clinical or pathological alterations were associated after chromium administration alone, despite its high levels in blood and tissue while cobalt–chromium and cobalt-treated rabbits showed clinical signs indicative of auditory and optic system toxicity. On histopathological examination, the animals showed severe retinal and cochlear ganglion cell depletion along with optic nerve damage and loss of sensory cochlear hair cells. Interestingly, the severity of the alterations was related to dosages and time of exposure. These data confirmed our previous observation of severe auditory and optic nerve toxicity in patients exposed to an abnormal release of cobalt and chromium from damaged hip prostheses. Moreover, we have identified the major element mediating neurotoxicity to be cobalt, although the molecular mechanisms mediating this toxicity still have to be defined.

Despite the toxicological risks to which humans and animals are exposed due to the transfer of toxic xenobiotic metabolites into milk of domestic animals, studies on the metabolizing mechanisms occurring in ruminant mammary gland are totally lacking. To investigate the possible biotransformation capabilities of a bovine mammary epithelial cell line (BME-UV1), monolayers were exposed to Aflatoxin B1 (AFB1 - 1.0 to 8.0 μM). Starting from 4 h of exposure, the hydroxylate metabolite Aflatoxin M1 (AFM1) was detected in media by high performance liquid chromatography. AFM1 concentration increased linearly with time for 36-48 h and the percent biotransformation of AFB1 (2 - 4 μM) at 48 h was about 12-14%. Parallel cytotoxicity assays (neutral red uptake-NRU and MTT assays) were performed to investigate the possible interference of AFB1 cytotoxitity with cellular metabolism. MTT assay (from 24 h of cell exposure) and NRU assay (from 16 h of cell exposure) showed time-dependent and time/concentration-dependent decrease of cell viability, respectively, being the former assay more successful to reveal cytotoxic effects (NRU: CC50 at 48h = 12.00±2.66 μM; MTT: CC50 at 72 h = 20.42±7.30 μM). The results suggest that BME-UV1 cells express metabolizing enzymes having catalytic activity, thus representing a potential in vitro model for studying biotransformation in bovine mammary gland.

Mesenchymal stromal cells (MSCs) isolated from human umbilical cord tissue (UCT) can be considered the perfect candidates for cell-based therapies and regenerative medicine. UCT-derived MSCs can be cryogenically stored in cell banks and expanded as needed for therapeutic uses.We developed a new method for UCT-MSC isolation, cryopreservation, and expansion, following all criteria required by a stem cell bank. UCT-MSCs were isolated either by manual dissociation (MM) or by a semiautomatic dissociation system (SAM). In both protocols UCTs were treated enzymatically using Type IV collagenase good manufacturing practices (GMP) graded and hyaluronidase (medicinal product). Isolated UCT-MSCs were cryopreserved and analyzed after thawing for phenotype; for proliferation rate; and for their osteogenic, adipogenic, and chondrogenic differentiation capabilities.We found that SAM reduced the time of tissue enzyme exposure and enabled us to obtain a homogeneous single-cell suspension deprived of tissue fragments. The isolated cells in both groups showed high expression of MSC markers CD105, CD73, and CD90 and similar differentiation capabilities, phenotype, and proliferation potential. Moreover, the final yield of MSCs was comparable between the two techniques.In this study, we have established a reliable and standardized protocol to isolate UCT-MSCs from UCT for cell banking purposes. Processing the whole umbilical tissue with GMP-graded enzymes using a semiautomatic dissociator allowed us to obtain a single-cell suspension product with a known number of isolated cells that can be cryopreserved right after isolation and thawed as needed for expansion and clinical use.

Background Multiple myeloma (MM) is a plasma cell malignancy with a multifaceted immune dysfunction. Indoleamine 2,3-dioxygenase 1 (IDO1) degrades tryptophan into kynurenine (KYN), which inhibits effector T cells and promote regulatory T-cell (Treg) differentiation. It is presently unknown whether MM cells express IDO1 and whether IDO1 activity correlates with immune system impairment. Methods We investigated IDO1 expression in 25 consecutive patients with symptomatic MM and in 7 patients with either monoclonal gammopathy of unknown significance (MGUS; n=3) or smoldering MM (SMM; n=4). IDO1-driven tryptophan breakdown was correlated with the release of hepatocyte growth factor (HGF) and with the frequency of Treg cells and NY-ESO-1-specific CD8+ T cells. Results KYN was increased in 75% of patients with symptomatic MM and correlated with the expansion of CD4+CD25+FoxP3+ Treg cells and the contraction of NY-ESO-1-specific CD8+ T cells. In vitro, primary MM cells promoted the differentiation of allogeneic CD4+ T cells into bona fide CD4+CD25hiFoxP3hi Treg cells and suppressed IFN-γ/IL-2 secretion, while preserving IL-4 and IL-10 production. Both Treg expansion and inhibition of Th1 differentiation by MM cells were reverted, at least in part, by d,l-1-methyl-tryptophan, a chemical inhibitor of IDO. Notably, HGF levels were higher within the BM microenvironment of patients with IDO+ myeloma disease compared with patients having IDO- MM. Mechanistically, the antagonism of MET receptor for HGF with SU11274, a MET inhibitor, prevented HGF-induced AKT phosphorylation in MM cells and translated into reduced IDO protein levels and functional activity. Conclusions These data suggest that IDO1 expression may contribute to immune suppression in patients with MM and possibly other HGF-producing cancers.

BACKGROUND: Human cord blood is a relevant source of CD133+ HPCs. Clinical-scale isolation of human umbi-lical cord blood (UCB) CD133+ HPCs using immunomag-netic microbeads and the CliniMACS clinical cell isolator is reported. CD133+ HPCs isolated after large-scale pro-cessing were functionally characterized. STUDY DESIGN AND METHODS: Closed disposable sets were used to process nine different samples of RBC-reduced UCB nucleated cells. In-vitro hematopoietic assays and human xenografts in NOD/SCID mice were performed to assess the functional properties of isolated CD133+ cells. Different mixtures of human cytokines were tested for the ability to expand nascent CD133+ HPCs. Furthermore, freshly isolated CD133+ cells were conditioned in culture medium specifically tested to support in-vitro myogenesis or osteogenesis. RESULTS: Isolation procedures yielded the recovery of an average of 2.53 × 106 CD133+ HPCs with a mean recovery of 96 percent (referred to as RBC-reduced samples) and a final sample purity of 82 percent. Purified CD133+ cells had high cloning efficiency, had relevant long-term activity, and were capable of repopulating irradiated NOD/SCID mice. In 10-day stroma-free cultures, a 2-fold and 8.3-fold expansion of colony-forming cells (CFCs) and extended long-term culture-initiating cells, respectively, was obtained. Freshly isolated CD133+ cells differen-tiated into large nucleated cells expressing either myosin D or osteopontin (as revealed by RT-PCR and immuno-cytochemistry), with a protein/mRNA expression compar-able to or even higher than that observed in UCB CD133– nucleated cells in identical culture conditions. CONCLUSION: Collectively, clinical-scale isolation of UCB CD133+ cells provides a relevant amount of primi-tive HPCs with high hematopoietic activity and in-vitro mesenchymal potential.

Granulocyte colony-stimulating factor (G-CSF) may affect T-cell homeostasis by multiple mechanisms, inducing polarization of cytokine secretion, inhibition of T-cell proliferation, and enhancement of T-cell apoptosis. We analyzed the production of interleukin-10 (IL-10) and transforming growth factor-beta1 (TGF-beta1) by T cells from healthy volunteer donors treated with recombinant human G-CSF. Highly purified CD4(+) T cells obtained before and after G-CSF administration (pre-G and post-G, respectively) were activated using the allogeneic mixed leukocyte reaction. Post-G CD4(+) T cells produced high levels of IL-10 but undetectable levels of IL-2 and IL-4, whereas the level of TGF-beta1 release was comparable to that of pre-G CD4(+) T cells. Notably, post-G CD4(+) T cells proliferated poorly in response to alloantigens and to recall antigens and suppressed the proliferation of autologous CD4(+) T cells in a cell contact-independent and an antigen-nonspecific manner. TGF-beta1 and IL-10 were not dispensable for post-G CD4(+) T cells to mediate suppression, as shown by neutralization studies. Compared with pre-G CD4(+) T cells, alloantigen-activated post-G CD4(+) T cells preferentially expressed markers associated with memory T cells, in conjunction with reduced levels of CD28 and CD62L. Collectively, these data demonstrate that CD4(+) T cells exposed to G-CSF in vivo acquire the properties of T regulatory (Tr) cells once triggered in vitro through the T-cell receptor, including a peculiar cytokine production profile (IL-10(++)TGF-beta1(+)IL-2(low/-)IL-4(low/-)), an intrinsic low proliferative capacity, and a contact-independent suppression of antigen-driven proliferation. Tr cells generated ex vivo after exposure to G-CSF might be clinically relevant for transplantation medicine and for the treatment of human immune-mediated diseases.

Objective The aim of this study was to evaluate the occurrence of T-cell spontaneous apoptosis (A spont ) and its modulation in vitro by the interleukin-2 receptor (IL-2R) γ-chain (γc)-signaling cytokine IL-15 in patients transplanted with autologous peripheral blood progenitor cells (PBPC) for hematologic malignancies. Materials and Methods Patients were examined on days 30–60, 60–90, and 90–120 after PBPC infusion. Dissipation of mitochondrial transmembrane potential, a hallmark of T-cell apoptosis, has been detected using the fluorescent probe 3,3′-dihexyloxacarbocyanine iodide, after short-term T-cell culture in the absence or presence of exogenous cytokines. Expression of Bcl-2 family members has been studied by flow cytometry and reverse transcriptase polymerase chain reaction. T-cell proliferative responses to recall antigens have been estimated in autologous mixed leukocyte cultures. Results A spont was seen in 45% ± 6% of CD4 + and 55% ± 6% of CD8 + T cells cultured in the absence of cytokines. Of interest, IL-15 and, to a lesser extent, its structural cousin IL-2 counteracted T-cell A spont by inhibiting the processing of caspase-3 and up-regulating Bcl-2 mRNA and protein levels. Cell division tracking confirmed that IL-15 did not rescue T cells from A spont by promoting proliferation but rather acted as a genuine survival factor. Addition of a γc-blocking antibody to cytokine-conditioned cultures abrogated both apoptosis inhibition and Bcl-2 induction by IL-15, suggesting involvement of the IL-2Rγc signal transduction pathway. Whereas cytokine-unprimed posttransplant T cells mounted inadequate responses to recall antigens, T cells conditioned with IL-15 expanded vigorously, indicating restoration of antigen-specific proliferation. Conclusions T cells recovering after autologous PBPC transplantation are highly susceptible to spontaneous apoptosis in vitro. This phenomenon can be counteracted by the γc-signaling cytokine IL-15. These findings suggest that IL-15 might be a promising immunomodulating agent to improve postgrafting T-cell function.

Whiteboards support face to face meetings by facilitating the sharing of ideas, focusing attention, and summarizing. However, at the end of the meeting participants desire some record of the information from the whiteboard. While there are whiteboards with built-in printers, they are expensive and relatively uncommon. We consider the capture of the information on a whiteboard with a mobile phone, improving the image quality with a cloud service, and sharing the results. This paper describes the algorithm for improving whiteboard image quality, the user experience for both a web widget and a smartphone application, and the necessary adaptations for providing this as a web service. The web widget, and mobile apps for both iPhone and Android are currently freely available, and have been used by more than 50,000 people.

Abstract 1476 Poster Board I-499 Background: Umbilical cord blood (UCB) is increasingly used as an alternative source of transplantable CD34+ haematopoietic stem cells (HSC) for neoplastic and non-neoplastic diseases. In addition to CD34-expressing 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 in the presence of a stromal cell support. IL-21 is a four-helix bundle cytokine released by activated CD4+ T cells and by NKT cells. 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. Methods: CD34−lineage− cells were maintained in liquid culture with 10−6M hydrocortisone, 20 ng/ml Flt3-L and 20 ng/ml SCF, with the addition of 50 ng/ml IL-15 and 20 ng/ml 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 the following parameters: a) cell surface phenotype; b) expression of molecular determinants of lymphoid/NK cell differentiation; c) secretion of IFN-γ, GM-CSF, TNF-α and CCL3/MIP-1α; d) cytolytic activity against NK-sensitive tumour cell targets and e) relative amount of Stat1 (Tyr701), Stat3 (Tyr705) and Stat5 (Tyr694) phosphorylation in response to IL-21. For all the above detailed experiments, control cultures were established with UCB-derived CD34+ HSC. Results: Freshly isolated CD34−lineage− cells stained negatively for stem cell-associated (CD34, CD133) and NK/lymphoid surface antigens (CD7, CD56, CD16, CD3, TCRαβ), and comprised 0.22% on average of UCB mononuclear cells (samples analyzed = 8). CD34−lineage− cells proliferated vigorously in response to IL-15 and IL-21 (average fold expansion at week +4 of culture = 42.5) but not to IL-21 alone, and up-regulated phosphorylated Stat1 and Stat3 proteins, in good agreement with previously published reports on the IL-21-induced activation of Stat signaling. CD34−lineage− cells expanded by IL-21 in combination with IL-15 acquired a peculiar lymphoid morphology with heavy cytoplasmic granules. When compared with CD34-derived NK cells, CD34−lineage− cells emerging from IL-15+IL-21-containing cultures expressed very low levels of CD16 and killer-cell immunoglobulin-like receptor (KIR), but high levels of CD56, NKG2D and IL-21 receptor, consistent with pseudo-mature NK cells. IL-21/IL-15-differentiated cells up-regulated mRNA signals for Bcl-2, GATA-3 and Id2, a master switch required for NK-cell development, and harboured un-rearranged TCRγ genes, suggesting that NK commitment under the experimental conditions here established occurs through a pathway that does not include TCR rearrangement. 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. Specifically, an average 65±11% of CD56+ NK cells differentiated with IL-15+IL-21 stained positively for CD107a in co-cultures established with NK-sensitive K562 cells. NK cell degranulation occurred at significantly lower levels in co-cultures containing K562 cells and IL-15-differentiated CD34−lineage− cells (mean percentage of CD107a+CD56+ NK cells equal to 35±6 at E:T ratio = 1; p < 0.01 compared with cultures containing IL-15+IL-21-matured NK cells), suggesting that IL-15 and IL-21 exerted synergistic effects on NK activity. Finally, NK cells differentiated from CD34+ HSC with either IL-15 alone or IL-15+IL-21 manifested a similar cytotoxic activity to that of cytokine-differentiated CD34−lineage− cells. Conclusions: This study suggests that considerable numbers of highly pure, lytic CD56+CD16−/+ NK cells for adoptive immunotherapy can be obtained from UCB CD34−lineage− cells using a serum-free, feeder cell-free culture system. The findings highlighted herein also shed some light into the developmental intermediates of NK cells that can be differentiated after the exposure of CD34−lineage− cells to IL-21. Disclosures: No relevant conflicts of interest to declare.

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.

Study design Observational Study. Objective To evaluate the effects of neurologic and non-neurologic factors on walking level and performance in chronic spinal cord lesion (SCL) patients. Summary of background data Walking is one of the primary goals of patients after a SCL. Several studies have demonstrated that different neurologic and non-neurologic factors can affect walking level and performance. However, in SCL age and muscle strength have always been considered the major determinants of walking. Methods Sixty-five patients with chronic SCL were included. Their demographic, neurologic status (ASIA standards), balance, and spasticity were recorded. Pearson and Spearman correlations were adopted to quantify the association between patients' characteristics and walking ability. The relationship between functional walking measures, Timed Up and Go, Six Minutes Walking Test (SMWT), Ten Meters Walking Test, and Walking Index for Spinal Cord Injury, and demographic and neurologic factors were measured by regression analyses. Results Strength, balance, spasticity, and age were strictly correlated with walking level and walking performance. They also were the best predictors of walking features. Conclusion Results confirm the recognized importance of age and upper and lower extremity strengths for walking after a SCL. They also highlight the role of 2 other factors, i.e., balance and spasticity, seldom considered as thoroughly in SCL.

Cancer stem cells have been isolated from several solid tumors including prostate, colon, liver, breast, and ovarian cancer. Stem cells isolated from nervous system and prostate express CD133 antigen, which is widely used to isolate hematopoietic stem and progenitor cells. The aims of this study were to investigate the expression of the CD133-1 and CD133-2 epitopes in primary ovarian tumors and to biologically characterize CD133+ovarian cancer cells, also according to clinicopathologic parameters. Tissue specimens were obtained at primary surgery from 41 ovarian carcinomas; eight normal ovaries and five benign ovarian tumors were also collected. Flow cytometry with monoclonal antibodies against CD133-1 and CD133-2 epitopes was employed. FACS (fluorescence activated cell sorting) analysis enabled the selection of CD133+cells, whose epithelial origin was confirmed by immunofluorescence analysis with monoclonal anti-cytokeratin 7. CD133+cells gave rise to a 4.7 ± 0.9-fold larger number of colonies than that documented in CD133−population (P< 0.001). Moreover, CD133+cells showed an enhanced proliferative potential compared to CD133−cells. The percentages of CD133-1- and CD133-2-expressing cells were significantly lower in normal ovaries/benign tumors with respect to those in ovarian carcinoma. Both the percentages of CD133-1- and CD133-2-expressing cells were significantly lower in omental metastases than in primary ovarian cancer (P= 0.009 and 0.007 for CD133-1- and CD133-2-expressing cells, respectively). There seems not to be any difference in the distribution of the percentage of CD133-1- and CD133-2-expressing cells according to clinicopathologic parameters and response to primary chemotherapy. CD133-1 and CD133-2 may be useful in order to select and enrich the population of CD133+ovarian tumor cells, which are characterized by a higher clonogenic efficiency and proliferative potential.

Objective To evaluate the effects of granulocyte colony-stimulating factor (G-CSF) on the in vitro sensitivity of acute myeloid leukemia (AML) cell lines and primary AML blast cells to gemtuzumab ozogamicin (GO). Materials and Methods AML cell lines and primary blasts from 10 patients with AML were first incubated for 72 hours in the presence of G-CSF (5 or 100 ng/mL) and then exposed to increasing concentrations of GO (1–1000 ng/mL) for an additional 72 hours. Results Pretreatment with G-CSF translated into significant enhancement of GO-induced cytotoxicity in the GO-sensitive HL-60 and NB-4 cells. Conversely, the response of GO-insensitive KG-1a, TF-1, and K562 cells was unaffected by in vitro priming with G-CSF. In vitro exposure to G-CSF augmented GO-induced apoptosis in 7 of 10 primary AML samples and rendered blast cells from three refractory patients sensitive to killing effect of GO. The G-CSF–induced increase of the cytocidal activity of GO was independent of effects on the cell cycle and on the expression levels of CD33 antigen. Of potential interest, G-CSF induced dose-dependent inhibition of P-glycoprotein (P-gp/ABCB1) function in the GO-sensitive HL-60 and NB-4 cells and in blasts from three patients with AML that we tested. Conclusion Collectively, our findings point to G-CSF as a potential sensitizing agent that can be exploited therapeutically to improve the clinical efficacy of GO.

We have recently demonstrated that G-CSF promotes the generation of human T regulatory (T(REG)) type 1 cells. In this study, we investigated whether the immunomodulatory effects of G-CSF might be mediated by DC. CD14(+) monocytes were cultured with serum collected after clinical administration of G-CSF (post-G), which contained high amounts of IL-10 and IFN-alpha. Similar to incompletely matured DC, monocytes nurtured with post-G serum acquired a DC-like morphology, expressed high levels of costimulatory molecules and HLA-DR, and exhibited diminished IL-12p70 release and poor allostimulatory capacity. Importantly, post-G DC-like cells were insensitive to maturation stimuli. As shown by neutralization studies, IFN-alpha and, even more pronounced, IL-10 contained in post-G serum inhibited IL-12p70 release by post-G DC-like cells. Furthermore, phenotypic and functional features of post-G DC-like cells were replicated by culturing post-G monocytes with exogenous IL-10 and IFN-alpha. Post-G DC-like cells promoted Ag-specific hyporesponsiveness in naive allogeneic CD4(+) T cells and orchestrated a T(REG) response that was dependent on secreted TGF-beta 1 and IL-10. Finally, neutralization of IL-10 and IFN-alpha contained in post-G serum translated into abrogation of the regulatory features of post-G DC-like cells. This novel mechanism of immune regulation effected by G-CSF might be therapeutically exploited for tolerance induction in autoimmune disorders.

Human cord blood is a relevant source of CD133+ HPCs. Clinical-scale isolation of human umbilical cord blood (UCB) CD133+ HPCs using immunomagnetic microbeads and the CliniMACS clinical cell isolator is reported. CD133+ HPCs isolated after large-scale processing were functionally characterized.Closed disposable sets were used to process nine different samples of RBC-reduced UCB nucleated cells. In-vitro hematopoietic assays and human xenografts in NOD/SCID mice were performed to assess the functional properties of isolated CD133+ cells. Different mixtures of human cytokines were tested for the ability to expand nascent CD133+ HPCs. Furthermore, freshly isolated CD133+ cells were conditioned in culture medium specifically tested to support in-vitro myogenesis or osteogenesis.Isolation procedures yielded the recovery of an average of 2.53 x 10(6) CD133+ HPCs with a mean recovery of 96 percent (referred to as RBC-reduced samples) and a final sample purity of 82 percent. Purified CD133+ cells had high cloning efficiency, had relevant long-term activity, and were capable of repopulating irradiated NOD/SCID mice. In 10-day stroma-free cultures, a 2-fold and 8.3-fold expansion of colony-forming cells (CFCs) and extended long-term culture-initiating cells, respectively, was obtained. Freshly isolated CD133+ cells differentiated into large nucleated cells expressing either myosin D or osteopontin (as revealed by RT-PCR and immuno-cytochemistry), with a protein/mRNA expression comparable to or even higher than that observed in UCB CD133- nucleated cells in identical culture conditions.Collectively, clinical-scale isolation of UCB CD133+ cells provides a relevant amount of primitive HPCs with high hematopoietic activity and in-vitro mesenchymal potential.

Background. A contributory effect of IDO1 to inflammatory autoimmune pathologies such as rheumatoid arthritis and systemic lupus erythematosus has been recently described. We aimed to address whether IDO1-driven kynurenine (KYN) production correlates with degree of inflammation and disease extent in systemic sclerosis (SSc). Methods. Peripheral blood was obtained from 27 consecutive patients (median age 62 years, range 22-78; median disease duration = 10 months, range 1-35) with either limited cutaneous SSc (n=17) or diffuse cutaneous SSc (n=10). In 11 of the 27 patients, CD19+ B cells were magnetically purified to estimate IDO1 mRNA levels by quantitative PCR. Results. High serum KYN in SSc (3.17±0.82 μM compared with 1.82±0.37 μM in healthy controls; p

Multiple myeloma (MM) is a plasma cell (PC) malignancy with a unique ability to evade immunosurveillance through the induction of APC dysfunction and the expansion of regulatory T cells (Treg). Hepatocyte growth factor (HGF) is elevated in MM and confers an unfavorable prognosis. We have previously shown that HGF induces the tryptophan (Trp)-degrading enzyme indoleamine 2,3-dioxygenase (IDO) in human monocytes. In this study, IDO-1 was detected in OPM-2, MOLP-8 and LP-1 MM cell lines but not in HUNS-1 cells. Kynurenine (KYN) production and Trp depletion were evidenced in supernatants of IDO-1+ MM cells, suggesting that IDO-1 may be functional. When challenged with exogenous HGF, IDO- HUNS-1 cells up-regulated IDO-1 mRNA 6-fold on average. IDO-1+ MM cells promoted the conversion of allogeneic CD4+CD25- T cells into bona fide CD4+FoxP3+ Tregs. We also detected IDO-1 expression in 6 out of 10 PC samples from patients with MM. Higher percentages of CD4+FoxP3+ Tregs were measured in IDO-1+ compared with IDO-1- MM. Intriguingly, serum KYN levels were higher in IDO-1+ compared with IDO-1- MM, suggesting that IDO-1 may mediate the in vivo expansion of Tregs through immunosuppressive KYN. Finally, serum HGF was over-represented in IDO-1+ compared with IDO-1- MM, providing indirect clues in favor of in vivo HGF effects on IDO-1 expression. Collectively, these data point to HGF as a potential inducer of IDO-1 in MM. Whether this will impact on the ability of the immune system to control disease progression remains to be prospectively investigated in larger cohorts of patients.

Avoidance of immunosurveillance is presently considered as a hallmark of cancer. Indoleamine 2,3-dioxygenase (IDO) is a cytosolic enzyme which metabolizes L-tryptophan to kynurenines and induces T cell suppression either directly on T cells, or by altering antigen presenting cell function and generating regulatory T cells (Treg). Recently, IDO expression by cancer cells and/or cancer-associated stromal cells has been correlated with the induction of immune dysfunction by human solid tumors, such as ovarian and colon cancer. It has been previously shown that IDO may be expressed by human acute myeloid leukemia, where it promotes the differentiation of Treg cells (Curti A et al. Blood2007;109:2871–7). Cyclooxygenase (COX) is the rate-limiting enzyme in the synthesis of prostaglandins and exists in two isoforms, COX-1 and COX-2. COX-2 is over-expressed by several tumor types and reportedly affects multiple pathways involved in tumorigenesis, including angiogenesis, invasion, and tumor-induced immune suppression. The present study aimed to determine whether COX-2 inhibitors may interfere with the IFN-γ-induced expression of IDO-1 in leukemia cells. The IDO-negative HL-60 acute myeloid leukemia cells, characterized by a CD34− CD33+CD119+ phenotype, were challenged with 100 ng/ml IFN-γ, a well-known inducer of IDO. Cytokine-treated HL-60 cells markedly up-regulated IDO-1 mRNA (~ 66-fold after 96 hours of culture) and protein and released a significant amount of kynurenines (12.58±2.66 μM after 72 hours and 19.21±9.8 μM after 96 hours of culture compared with 1.35±0.36 μM and 1.31±0.42 μM from untreated HL-60 cells; p =0.0004). This was associated with an average 35% depletion of tryptophan from culture supernatants. Not unexpectedly, IFN-γ-treated HL-60 cells promoted the in vitro conversion of naïve allogeneic CD4+CD25− T cells into bona fide CD4+CD25+FoxP3+ Treg cells. Phosphorylated signal transducer and activator of transcription (STAT)-1 could be detected in HL-60 cells starting from 48 hours of IFN-γ treatment, but not in untreated HL-60 cells, and its expression kinetics and relative amount closely paralleled those of IDO-1. IFN-γ strongly induced COX-2 mRNA (average fold-induction equal to 17.7±1.37 compared with untreated HL-60 cells) and protein. Conversely, IFN-γ exerted no appreciable effects on the expression levels of the tryptophan transporter molecule (LAT-1/CD98 receptor complex) on HL-60 leukemia cells. The addition of 100 μM nimesulide, a preferential COX-2 inhibitor, to HL-60 cells that were pre-treated with IFN-γ as above detailed translated into an average 54% reduction of kynurenines in culture supernatants (18.78±4.02 μM in the absence of nimesulide compared with 8.75±3.26 μM in cultures established with the drug; p = 0.0082). Superimposable results in terms of kynurenine production and tryptophan depletion were recorded in cultures of HL-60 cells that were simultaneously established with IFN-γ and nimesulide. Exposure to nimesulide attenuated mRNA signals for IDO-1, suggesting that the overall inhibition of IDO activity leading to suppressed kynurenine synthesis might also be attributed to the reduction of IDO-1 gene transcription. Interestingly, nimesulide diminished the expression of phosphorylated STAT-1 in HL-60 cells, indicating the involvement of this signaling pathway in the regulation of IDO-1 expression. In a further set of experiments, aspirin was added for 24 hours at 1 mM (final concentration) to IFN-γ-treated HL-60 cells in order to investigate its effects on IDO expression and function. In good agreement with the above results with nimesulide, aspirin promoted an average 65% inhibition of kynurenine release from IFN-γ-treated HL-60 cells and abrogated tryptophan consumption from culture supernatants. Collectively, these data suggest that COX-2 inhibition might be exploited as a valuable strategy to interfere with IDO-mediated tryptophan catabolism and hopefully to interfere with the tumor-induced immune dysfunction.

CD34−lineage− cells encompass an average 0.16% of human umbilical cord blood (UCB) mononuclear cells. We have previously shown that IL-15 promotes the stromal cell-dependent differentiation of UCB CD34−lineage− cells into bipotent T/NK progenitor cells. IL-21, a CD4+ T-cell-derived IL-2 receptor γ-chain signalling cytokine, is a crucial regulator of NK cell development and function. IL-21 increases the generation of mature NK cells from CD34+ haematopoietic stem cells (HSC) in combination with IL-15, IL-7, SCF and Flt3 ligand. To date, it has not been established whether IL-21 might replace the stromal cell requirements and synergise with IL-15 in the induction of NK cell differentiation of CD34−lineage− cells. In the present study, highly purified CD34−lineage− cells from human UCB were cultured in medium containing IL-15, SCF and Flt3 ligand, either in the absence or presence of IL-21. Cells were analysed at different time intervals for surface expression of NK cell-associated antigens (CD56, CD16, NKG2A/CD94 inhibitory receptor, CD244 and NKG2D activating receptors, killer-cell immunoglobulin-like receptors, NKp46 triggering receptor), mRNA signals for NK-specific transcription factors (inhibitor of DNA binding protein 2 or ID2) and bcl-2 family members, T-cell-receptor (TCR)γ rearrangement status, IFN-γ release, and lytic activity against NK-sensitive tumour targets. On day +30 of culture in the presence of IL-15 and IL-21, CD34−lineage− cells were expanded by 35-fold on average, compared with 21-fold in cultures nurtured with IL-15 alone. IL-21 per se was incapable of activating the proliferation of CD34−lineage− cells or promote the emergence of NK cell-associated surface markers (see below). After 30 days of culture in the presence of IL-21, 80–90% of the developing NK cells exhibited a CD56brightCD16neg phenotype. Consistent with the antigen expression profile previously assigned to pseudomature NK cells, IL-21-differentiated NK cells (IL-21NKs) stained negatively for killer-cell immunoglobulin-like receptors (CD158a and CD158b). Whereas NKG2A/CD94 expression on IL-21NKs mirrored that on IL-15-differentiated NK cells, NKG2D could be detected on an average 65% of IL-21NKs compared with 10% of cells maintained with IL-15. In sharp contrast to IL-15 alone, the combination of IL-21 and IL-15 markedly up-regulated mRNA signals for bcl-2, bcl-xL, TGF-β, GATA-3 and, perhaps more importantly, ID2, a master switch required for NK-cell development. At variance with IL-15-differentiated NK cells, IL-21NKs harboured unrearranged TCRγ genes, secreted copious amounts of IFN-γ in culture supernatants and were capable of lysing NK-sensitive tumour cell targets after pre-activation with exogenous IL-2. Figure Figure The present study establishes a novel role for IL-21 in the expansion and differentiation of pseudomature lytic NK cells, primarily in a synergistic context with IL-15. IL-21 might represent a valuable strategy to expand lytic NK cells from UCB CD34−lineage− cells for immunotherapy purposes.

Dendritic cells (DCs) are highly specialized antigen presenting cells (APCs) with unique capacity to fully activate and induce clonal expansion of naive and memory T cells. Recently, DCs have been implicated in the maintenance of antigen (Ag)-specific unresponsiveness or tolerance through the induction of regulatory T (Treg) cells. Several hematopoietic growth factors, including interleukin-10 (IL-10), transforming growth factor-β1 (TGF-β1) and granulocyte colony-stimulating factor (G-CSF), might promote the differentiation of regulatory dendritic cells (DCs) in vivo and/or in vitro. Hepatocyte growth factor (HGF) is a pleiotropic cytokine and its effects on human DC differentiation and function have not been investigated. Monocytes cultured with HGF (HGF-DCs) differentiated into accessory cells with DC features, released low amounts of IL-12p70 and up-regulated IL-10 both at the mRNA and at the protein level. HGF-DCs displayed a CD14+CD1a−costimulationlow phenotype, and expressed the inhibitory receptor ILT3. Upon activation with HGF-DCs, allogeneic CD4+CD25− T cells up-regulated CD25 and the Treg-associated transcription factor FoxP3, proliferated poorly and released high levels of IL-10 but trace amounts of IL-2 and IFN-≤ . Interestingly, blockade of surface ILT3 on HGF-DCs or neutralization of secreted IL-10 translated into partial restoration of T-cell proliferation. Addition of exogenous IL-2 to CD4+ T cells initially primed with HGF-DCs was not associated with the enhancement of T cell proliferation. Secondary stimulation of HGF-DC-primed CD4+ T cells with immunogenic DCs differentiated with GM-CSF and IL-4 from monocytes of the same donor resulted in optimal T-cell proliferation. Interestingly, HGF-DC-primed CD4+ T cells significantly inhibited the proliferation of naive CD4+CD25− T cells in a cell contact-dependent manner, as shown by co-culture experiments with transwell inserts. Figure Figure Finally, DNA microarray analysis revealed a unique gene expression profile of HGF-activated monocytes. Specifically, HGF-DCs over-expressed a set of 217 genes implicated in cellular metabolism, signal transduction, cell adhesion, cell-to-cell signaling, inflammation and immune response. Genes up-regulated by HGF in freshly isolated monocytes are categorized according to their biological function Defense response 19 Cell adhesion and signaling 12 Signal transduction 32 Transcription regulation 17 Metabolism 35 Protein synthesis and degradation 24 Cytoskeletal organization 7 Transport 16 Chemotaxis 9 Cell proliferation and differentiation 6 Diverse physiological functions 9 Response to stress 9 Cell growth 4 Cell cycle 4 Morphogenesis 13 Unclassified 31 Of interest, HGF-DCs also upregulated mRNA signals for genes involved in tryptophan catabolisms, e.g., indoleamine 2,3-dioxygenase (IDO). Collectively, our findings point to a novel role for HGF in the regulation of monocyte/DC functions. From a therapeutic standpoint, HGF might represent an attractive target for immunotherapy.

The hemopoietic stem cell (HSC) compartment encompasses cell subsets with heterogeneous proliferative and developmental potential. Numerous CD34(-) cell subsets that might reside at an earlier stage of differentiation than CD34(+) HSCs have been described and characterized within human umbilical cord blood (UCB). We identified a novel subpopulation of CD34(-)CD133(-)CD7(-)CD45(dim)lineage (lin)(-) HSCs contained within human UCB that were endowed with low but measurable extended long-term culture-initiating cell activity. Exposure of CD34(-)CD133(-)CD7(-)CD45(dim)lin(-) HSCs to stem cell factor preserved cell viability and was associated with the following: 1) concordant expression of the stem cell-associated Ags CD34 and CD133, 2) generation of CFU-granulocyte-macrophage, burst-forming unit erythroid, and megakaryocytic aggregates, 3) significant extended long-term culture-initiating cell activity, and 4) up-regulation of mRNA signals for myeloperoxidase. At variance with CD34(+)lin(-) cells, CD34(-)CD133(-)CD7(-)CD45(dim)lin(-) HSCs maintained with IL-15, but not with IL-2 or IL-7, proliferated vigorously and differentiated into a homogeneous population of CD7(+)CD45(bright)CD25(+)CD44(+) lymphoid progenitors with high expression of the T cell-associated transcription factor GATA-3. Although they harbored nonclonally rearranged TCRgamma genes, IL-15-primed CD34(-)CD133(-)CD7(-)CD45(dim)lin(-) HSCs failed to achieve full maturation, as manifested in their CD3(-)TCRalphabeta(-)gammadelta(-) phenotype. Conversely, culture on stromal cells supplemented with IL-15 was associated with the acquisition of phenotypic and functional features of NK cells. Collectively, CD34(-)CD133(-)CD7(-)CD45(dim)lin(-) HSCs from human UCB displayed an exquisite sensitivity to IL-15 and differentiated into lymphoid/NK cells. Whether the transplantation of CD34(-)lin(-) HSCs possessing T/NK cell differentiation potential may impact on immunological reconstitution and control of minimal residual disease after HSC transplantation for autoimmune or malignant diseases remains to be determined.

Background Pegylated 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. Methods Twelve 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. Results Pegfilgrastim 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. Conclusions Pegfilgrastim 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.