A new standardized clinical-grade protocol for banking human umbilical cord tissue cells.

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.

Cyclooxygenase-2 (COX-2) inhibition constrains indoleamine 2,3-dioxygenase 1 (IDO1) activity in acute myeloid leukaemia cells.

Indoleamine 2,3-dioxygenase 1 (IDO1) metabolizes L-tryptophan to kynurenines (KYN), inducing T-cell suppression either directly or by altering antigen-presenting-cell function. Cyclooxygenase (COX)-2, the rate-limiting enzyme in the synthesis of prostaglandins, is over-expressed by several tumours. We aimed at determining whether COX-2 inhibitors down-regulate the IFN-g-induced expression of IDO1 in acute myeloid leukaemia (AML) cells. IFN-γ at 100 ng/mL up-regulated COX-2 and IDO1 in HL-60 AML cells, both at mRNA and protein level. The increased COX-2 and IDO1 expression correlated with heightened production of prostaglandin (PG)E2 and kynurenines, respectively. Nimesulide, a preferential COX-2 inhibitor, down-regulated IDO1 mRNA/protein and attenuated kynurenine synthesis, suggesting that overall IDO inhibition resulted both from reduced IDO1 gene transcription and from inhibited IDO1 catalytic activity. From a functional standpoint, IFN-g-challenged HL-60 cells promoted the in vitro conversion of allogeneic CD4⁺CD25⁻ T cells into bona fide CD4⁺CD25⁺FoxP3⁺ regulatory T cells, an effect that was significantly reduced by treatment of IFN-γ-activated HL-60 cells with nimesulide. Overall, these data point to COX-2 inhibition as a potential strategy to be pursued with the aim at circumventing leukaemia-induced, IDO-mediated immune dysfunction.

Indoleamine 2,3-dioxygenase 1 (IDO1) activity correlates with immune system abnormalities in multiple myeloma.

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(+)CD25(hi)FoxP3(hi) 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.

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

BACKGROUND: Cytokine-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. METHODS: Peripheral 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. RESULTS: CIK 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. CONCLUSIONS: TG 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.

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-ß1 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.

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

BACKGROUND: Umbilical 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. RESULTS: CD34-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-gamma, GM-CSF, TNF-alpha and CCL3/MIP-1alpha, 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 TCRgamma genes. From a functional standpoint, IL-21/IL-15-treated cells secreted copious amounts of IFN-gamma, GM-CSF and CCL3/MIP-1alpha, and expressed cell surface CD107a upon contact with NK-sensitive tumour targets, a measure of exocytosis of NK secretory granules. CONCLUSION: This 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.

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 GalNAcalpha1-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. STUDY DESIGN AND METHODS: Nine UCB units were subjected to sequential red cell removal, freezing, and postthawing CD133+ HSC immunoselection by a clinical-grade, CE-approved, magnetic apparatus and microbead-coated anti-CD133 monoclonal antibody. Selected UCB CD133+ cells were cultured in vitro in medium supporting either endothelial or cardiomyocytic differentiation in parallel experiments. RESULTS: Immunoselection allowed recovery of 79 percent of initial CD133+ cells with a CD133+ cell purity of 81 percent, on average. Parallel cultures showed the appearance of endothelial markers (VE-cadherin, CD146, and KDR and bright expression of CD105), morphofunctional features of endothelium in endothelial-supporting cultures, of cardiac muscle proteins (troponin I and myosin ventricular heavy chain alpha/beta; MYHC) and specific gene expression (GATA4, NKX2.5, troponin I, and MYHC) in cardiomyocyte-oriented cultures. CONCLUSIONS: The appearance of both endothelial- and cardiomyocyte-like cells from parallel cultures of frozen-thawed-immunoselected UCB CD133+ cells by a clinical-grade method and previously reported data on lack of major signs of rejection of these cells in immunocompetent rats subjected to experimental liver damage suggest a possible role of these allogeneic HSCs in cell therapies designed for regenerative treatments of ischemic diseases of human myocardium.

Hepatocyte growth factor favors monocyte differentiation into regulatory interleukin (IL)-10++IL-12low/neg accessory cells with dendritic-cell features.

Several hematopoietic growth factors, including interleukin-10 (IL-10) and transforming growth factor-beta1 (TGF-beta1), promote the differentiation of tolerogenic dendritic cells (DCs). Hepatocyte growth factor (HGF) is a pleiotropic cytokine whose effects on human DC differentiation and function have not been investigated. Monocytes cultured with HGF (HGFMo) differentiated into accessory cells with DC-like morphology, released low amounts of IL-12p70 and up-regulated IL-10 both at the mRNA and at the protein level. Upon activation with HGFMo, allogeneic CD4+CD25- T cells expressed the T regulatory (Treg)-associated transcription factor FoxP3, proliferated poorly, and released high levels of IL-10. Interestingly, blockade of surface immunoglobulin-like transcript 3 (ILT3) on HGFMo or neutralization of secreted IL-10 translated into partial restoration of T-cell proliferation. Secondary stimulation of HGFMo-primed CD4+ T cells with immunogenic DCs differentiated with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 from monocytes of the same donor resulted in measurable T-cell proliferation. HGFMo-primed CD4+ T cells significantly inhibited the proliferation of naive CD4+CD25- T cells in a cell-contact-dependent manner. Finally, DNA microarray analysis revealed a unique gene-expression profile of HGF-activated monocytes. Collectively, our findings point to a novel role for HGF in the regulation of monocyte/DC functions that might be exploited therapeutically.

Granulocyte colony-stimulating factor enhances the in vitro cytotoxicity of gemtuzumab ozogamicin against acute myeloid leukemia cell lines and primary blast cells.

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 (1-1,000 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.

Clinical isolation and functional characterization of cord blood CD133+ hematopoietic progenitor cells.

BACKGROUND: 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. 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 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. CONCLUSION: Collectively, clinical-scale isolation of UCB CD133+ cells provides a relevant amount of primitive HPCs with high hematopoietic activity and in-vitro mesenchymal potential.

Identification of a novel subpopulation of human cord blood CD34-CD133-CD7-CD45+lineage- cells capable of lymphoid/NK cell differentiation after in vitro exposure to IL-15.

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.

Role for granulocyte colony-stimulating factor in the generation of human T regulatory type 1 cells.

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.