Nonadherent Fraction Research Articles

Two Distinct Populations of Enriched Non Mobilized Peripheral Blood Mononuclear Cells (PBMNCs) with Different Functional Capacities

Pluripotent stem cells are valuable sources for transplantation and tissue repair. The bone-marrow, umbilical cord blood and G-CSF mobilized peripheral blood are the main sources for adult stem cells. Non-mobilized peripheral blood contains mostly committed cells but recent studies suggest the presence of early progenitor and stem cells as well. Here we aim to develop a method to enrich and recover functional progenitor cell populations from non-mobilized peripheral blood. The ex-vivo enriched non-mobilized PBMNCs were tested by FACS analysis of specific cell surface markers, and by functional analysis of differentiation into different cells lineages. Non mobilized PBMNCs obtained from consenting healthy donors (n=18) cultured for 7 days in a defined cytokine cocktail media were analyzed by FACS using CD14, CD31, CD34, CD45, CD90, CD105, CD117 and CD133 and compare to non-manipulated PBMNCs. The enriched cells were also tested for their differentiation capacity into hematopoietic, endothelial and mesenchymal cells lineages compared to non-manipulated PBMNCs. We found that the enriched PBMNCs resulted in two distinct subpopulations, adherent and non adherent, that present different cell surface markers and have different differentiation capacities. Cell surface markers analysis showed that adherent cells possess high percentage of CD14 (28.1 ±6.13, 11.8 ± 3.9), CD90 (4.24±0.94, 1.53 ± 0.28), CD105 (42.19±8.42, 7.96 ± 4.8), CD117 (9.89±5.99, 1.75± 0.8) expression and reduction of CD45 (46.3±8.14, 62.74± 9.6), CD31 (5.9±1.9, 15.34 ± 4.9) and CD34 (0.18±0.03, 0.62 ±0.3) compare to non-manipulated PBMNCs, respectively. On the other hand, the non-adherent sub-population expresses more CD34 (1.2±0.02, 0.16±0.02), KDR (3.62±0.82, 0.49±0.2), CD105 (21.62±1.85, 7.96 ± 4.8) and CD45 (88.7±0.51, 62.74± 9.6) compared to non-manipulated PBMNCs, respectively. The adherent cells subpopulation showed higher differentiation potential into endothelial (116+23.1 EC colonies/106 cells), mesenchymal (400±53.9 CFU-F/106 cells) lineages compare to non-adherent cells (4.9±1.7 EC colonies/106 cells, 200±29.5 CFU-F/106 cells) and compare to non-manipulated PBMNCs (11.4±1.4 EC colonies/106 cells and 2.5±0.08 CFU-F/106 cells). The non-adherent subpopulations showed higher differentiation potential into hematopoietic colonies (445±75 CFU/106 cells) compared to the adherent sub-population (41.2±24.8 CFU/106 cells), and compared to the non-manipulated PBMNCs (373±39.7 CFU/106 cells) of total colony numbers/106 cells. All results (n=18) are presented as (mean ± SE). In summary, our ex-vivo enrichment methodology yields two different subpopulations with enriched hematological lineage in the non-adherent fraction and enriched endothelial and mesenchymal lineages in the adherent fraction. The ability to obtain enriched populations of endothelial, mesenchymal and hematopoietic progenitors from non mobilized peripheral blood cells may have an important clinical application.

Vascular morphogenesis by adult bone marrow progenitor cells in three-dimensional fibrin matrices

The neovascularization of tissues is accomplished by two distinct processes: de novo formation of blood vessels through the assembly of progenitor cells during early prenatal development (vasculogenesis), and expansion of a pre-existing vascular network by endothelial cell sprouting (angiogenesis), the main mechanism of blood vessel growth in postnatal life. Evidence exists that adult bone marrow (BM)-derived progenitor cells can contribute to the formation of new vessels by their incorporation into sites of active angiogenesis. Aim of this study was to investigate the in vitro self-organizing capacity of human BM mononuclear cells (BMMNC) to induce vascular morphogenesis in a three-dimensional (3D) matrix environment in the absence of pre-existing vessels. Whole BMMNC as well as the adherent and non-adherent fractions of BMMNC were embedded in fibrin gels and cultured for 3–4 weeks without additional growth factors. The expression of hematopoietic-, endothelial-, smooth muscle lineage, and stem cell markers was analyzed by immunohistochemistry and confocal laser-scanning microscopy. The culture of unselected BMMNC in 3D fibrin matrices led to the formation of cell clusters expressing the endothelial progenitor cell (EPC) markers CD133, CD34, vascular endothelial growth factor receptor (VEGFR)-2, and c-kit, with stellar shaped spreading of peripheral elongated cells forming tube-like structures with increasing complexity over time. Cluster formation was dependent on the presence of both adherent and non-adherent BMMNC without the requirement of external growth factors. Developed vascular structures expressed the endothelial markers CD34, VEGFR-2, CD31, von Willebrand Factor (vWF), and podocalyxin, showed basement-membrane-lined lumina containing CD45 + cells and were surrounded by α-smooth muscle actin (SMA) expressing mural cells. Our data demonstrate that adult human BM progenitor cells can induce a dynamic self organization process to create vascular structures within avascular 3D fibrin matrices suggesting a possible alternative mechanism of adult vascular development without involvement of pre-existing vascular structures.

Phenotypic characterization of two cell populations involved in the acquisition of suppressor activity by cultured spleen cells from Mycobacterium lepraemurium-infected mice.

The impairment of cellular immunity in mice infected with Mycobacterium lepraemurium was shown to correlate with the development of suppressor cells. We have previously reported that before suppressor activity is detectable in freshly harvested cell suspensions, suppressor cell precursors accumulate in the spleen of infected mice. Upon overnight culture in the presence of a regulatory cell subset, these precursor cells acquire the capacity to impair the concanavalin A (Con A)-induced proliferation of normal spleen cells. The purpose of this study was to determine the phenotype of the cells involved in this phenomenon. This was done by following the development of suppressor activity in spleen cell suspensions depleted of defined cell subsets of the adherent or the non-adherent cell fractions with selected MoAbs and immunomagnetic beads or by in vivo treatment. Our results indicate that the acquisition of suppressor activity requires the interaction of Ia+CD11b+Fc gamma R+IgG- asialo GM1- adherent cells with Thy1-CD4-CD8-IgG-Ia- asialo GM1-Fc gamma R+CD11b+ non-adherent cells. It is also shown that the development of suppressor activity is impaired by preventing cell-cell contact between these two cell subsets through coculture in 'Transwell chambers'. These observations support the conclusion that the in vitro acquisition of suppressor activity is a consequence of the maturation of suppressor cell precursors of the monocytic lineage induced by a receptor-ligand type interaction with a non-adherent cell subset that is clearly distinct from mature T, B and natural killer (NK) cells.

Tumor Suppressor Protein p53 Regulates Megakaryocytic Polyploidization and Apoptosis

The molecular mechanisms underlying differentiation of hematopoietic stem cells into megakaryocytes are poorly understood. Tumor suppressor protein p53 can act as a transcription factor affecting both cell cycle control and apoptosis, and we have previously shown that p53 is activated during terminal megakaryocytic (Mk) differentiation of the CHRF-288-11 (CHRF) cell line. Here, we use RNA interference to reduce p53 expression in CHRF cells and show that reduced p53 activity leads to a greater fraction of polyploid cells, higher mean and maximum ploidy, accelerated DNA synthesis, and delayed apoptosis and cell death upon phorbol 12-myristate 13-acetate-induced Mk differentiation. In contrast, reduced p53 expression did not affect the ploidy or DNA synthesis of CHRF cells in the absence of phorbol 12-myristate 13-acetate stimulation. Furthermore, primary Mk cells from cultures initiated with p53-null mouse bone marrow mononuclear cells displayed higher ploidy compared with wild-type controls. Quantitative reverse transcription-PCR analysis of p53-knockdown CHRF cells, compared with the "scrambled" control CHRF cells, revealed that six known transcriptional targets of p53 (BBC3, BAX, TP53I3, TP53INP1, MDM2, and P21) were down-regulated, whereas BCL2 expression, which is known to be negatively affected by p53, was up-regulated. These studies show that the functional role of the intrinsic activation of p53 during Mk differentiation is to control polyploidization and the transition to endomitosis by impeding cell cycling and promoting apoptosis.

Vaccination strategy to target lysyl oxidase-like 4 in dendritic cell based immunotherapy for head and neck cancer

Overexpression of lysyl oxidase (LOX) is associated with the invasive potential of metastatic breast and head and neck cancer (HNC) cells and reduced metastasis-free and overall survival. Recently, we have demonstrated up-regulation of a new member of the LOX family, lysyl oxidase-like 4 (LOXL4), in invasive HNC revealed a significant correlation between LOXL4 expression and local lymph node metastases and higher tumour stages. The objective of this study was to examine whether cellular LOXL4 may provide an effective target for cell-meditated immunotherapy in invasive tumours associated with LOXL4 overexpression. As a feasibility study we expressed LOXL4 mRNA in immature dendritic cells derived from human peripheral blood mononuclear cells (PBMC). LOXL4 protein expression was ascertained using Western blotting and immunocytochemistry with polyclonal rabbit anti-LOXL4 antibody. The successfully transfected immature dendritic cells (DCs) were induced to mature with GM-CSF, IL-4, IL-1beta, TNF-alpha, IL-6, and PGE2, and then used to stimulate T cell enriched non-adherent fraction of PBMC. LOXL4 specific T cell stimulation induced cytotoxic T lymphocyte (CTL) response was monitored using IFN-gamma secretion from the non-adherent PBMC fraction exposed to mature, LOXL4 transfected DCs acting as the antigen presenting target cells. LOXL4-DC stimulated T cells produced higher IFN-gamma secretion compared to unstimulated T cells and T cells stimulated with untransfected DCs, in the presence of the pan-DR-epitope (PADRE). These initial results demonstrated the potential for LOXL4-transfected DCs to serve as efficient tumour vaccine and support their suitability as a vaccination strategy applicable to cancer patients with tumour specific up-regulation of LOXL4.

Adipogenesis in Nonadherent and Adherent Bone Marrow Stem Cells Grown in Fibrin Gel and in the Presence of Adult Plasma

Bone marrow-derived mesenchymal stem cells (i.e., adherent cells) are known to differentiate into fat tissue in the presence of adipogenic supplements in cultures. Induction of adipogenesis has not been investigated within the nonadherent cell fraction that includes predominantly hematopoietic cells. In the present study, murine nonadherent bone marrow-derived stem cells (96% CD45+ cells) were seeded and then grown in fibrin gel to form cell clusters in which most cells were positive to DiI-acetylated low-density lipoprotein uptake. Amongst different culture media supplemented either in fetal bovine serum, horse serum, murine plasma, human plasma or adipogenic supplements, a subpopulation of nonadherent stem cells within clusters differentiated into adipocytes, specifically in the presence of adult syngeneic plasma. This was confirmed by the observation and quantification of oil red O-positive cells, the measurement of glycerol-3-phosphate dehydrogenase activity and peroxisome proliferator-activated receptor-γ mRNA expression. Similarly, adipogenesis was also observed in the presence of murine plasma with adherent mesenchymal stem cells and 3T3-L1 preadipocytes which were grown either in monolayer plastic cultures or in fibrin gel. Thus, it is possible that nonadherent cells, once in a 3-dimensional environment, can further differentiate towards adipogenesis.

N-Cadherin and Cadherin-11 Play Vital Roles in the Cell-Cell Contact between Hematopoietic Progenitor Cells and Mesenchymal Stromal Cells.

Self renewal and differentiation of hematopoietic progenitor cells (HPC) are regulated by the microenvironment of the bone marrow. As an in vitro model system, mesenchymal stromal cells (MSC) provide a supportive cellular microenvironment for maintaining primitive function of HPC. It has been postulated that direct cell-cell interaction is crucial for maintenance of “stemness”. Human HPC were co-cultured with MSC from human bone marrow and subsequently separated into an adherent and a non-adherent fraction. HPC subsets with higher self-renewing capacity demonstrated significantly higher adhesion to MSC (CD34+vs. CD34−, CD34+/CD38−vs. CD34+/CD38+, slow dividing fraction vs. fast dividing fraction). Long-term culture-initiating cell (LTC-IC) frequency was higher in the adherent fraction than in the non-adherent fraction of CD34+ cells. Microarray analysis (Affymetrix, U133_Plus_2.0) revealed that differentially expressed genes coding for adhesion proteins were highly up-regulated in the adherent fraction of CD34+ cells. These genes included VCAM1, connexin 43 and cadherin-11. Furthermore, we have compared the supportive potential of different feeder layer preparations. Human MSC were isolated from bone marrow (BM), from adipose tissue (AT) and umbilical cord blood (CB). The ability to maintain LTC-IC and a primitive CD34+CD38− immunophenotype was significantly higher for MSC derived from BM and CB compared to those from AT. These results were in line with higher adhesion of HPC to BM-MSC and CB-MSC in comparison to AT-MSC. Analysis of the cytokine production of MSC preparations by antibody arrays, ELISA and by a cytometric bead array showed that albeit there were significant differences in the chemokine secretion profiles of the aforementioned MSC preparations, there was no relationship to their potentials in maintaining primitive function of HPC. Global gene expression profiles of MSC preparations showed that adhesion proteins including N-cadherin, cadherin-11, VCAM1, NCAM1 and integrins were highly expressed in MSC preparations derived from BM and CB. Western blot analysis confirmed higher protein expression of N-cadherin and cadherin-11 in BM-MSC compared to AT-MSC and CB-MSC. Fluorescent microscopic analysis revealed that N-cadherin is located at the cell-cell contacts between HPC and MSC. Expression of N-cadherin or cadherin-11 was efficiently knocked down in MSC feeder layer using siRNA. This effect was verified by Western blot analysis and it lasted for up to seven days. Adhesion of HPC was significantly reduced on MSC that have been treated by siRNAs for N-cadherin and cadherin-11 whereas siRNA for MAPK did not affect cell-cell interaction. Similarly, a blocking functional antibody for N-cadherin reduced significantly the adhesion of HPC to MSC. MSC provide a microenvironment which supports the maintenance of primitive function of HPC. Our results indicated that direct cell-cell interaction mediated by N-cadherin and cadherin-11 plays a central role in this interaction of HPC with their cellular microenvironment.

Adhesion of hematopoietic progenitor cells to human mesenchymal stem cells as a model for cell−cell interaction

The significant role of direct contact between hematopoietic progenitor cells (HPC) and the cellular microenvironment for maintaining "stemness" has been demonstrated. Human mesenchymal stem cell (MSC) feeder layers represent a surrogate model for this interaction. Specific adhesion molecules are responsible for this cell-cell contact. To define cell-cell contact between HPC and MSC, we have studied adhesive interaction of various fractions of HPC by using a novel assay based on gravitational force upon inversion. Adherent and nonadherent cells were separated and further analyzed with regard to gene expression and long-term hematopoietic culture initiating cell (LTC-IC) frequency. HPC subsets with higher self-renewing capacity demonstrated significantly higher adherence to human MSC (CD34(+) vs CD34(-), CD34(+)/CD38(-) vs CD34(+)/CD38(+), slow dividing fraction vs fast dividing fraction). LTC-IC frequency was significantly higher in the adherent fraction than in the nonadherent fraction. Furthermore, genes coding for adhesion proteins and extracellular matrix were higher expressed in the adherent subsets of CD34(+) cells (fibronectin 1, cadherin 11, vascular cell adhesion molecule-1, connexin 43, integrin beta-like 1, and TGFBI). In this study we have demonstrated that primitive subsets of HPC have higher affinity to human MSC. The essential role of specific junction proteins for stabilization of cell-cell contact is indicated by their significant higher expression.

Production of IL-7 is increased in ovariectomized mice, but not RANKL mRNA expression by osteoblasts/stromal cells in bone, and IL-7 enhances generation of osteoclast precursors in vitro

Osteoclastogenic cytokines produced by T and B lineage cells and interleukin (IL)-7-induced expansion of the pool size of osteoclast precursors have been suggested to play an important role in acceleration of osteoclastogenesis induced by estrogen deficiency. However, the contribution of increased RANKL produced by osteoblasts/stromal cells to increase osteoclastogenesis in a mouse model of estrogen-deficient osteoporosis and in vitro effects of IL-7 on osteoclast precursor generation remain controversial. Thus, we investigated the effect of ovariectomy (OVX) of mice on production of RANKL, osteoprotegerin (OPG), and IL-7 in bone and the effect of IL-7 on osteoclast precursor generation in vitro. OVX did not significantly stimulate mRNA expressions of RANKL and OPG in whole femurs. Because the epiphysis, but not the femoral shaft (diaphysis) or bone marrow, is the main site of osteoclastogenesis, it is important to specifically analyze mRNA expression by osteoblasts/stromal cells at these parts of the femur. Therefore, we isolated RNA from bone marrow cell-free epiphysis, diaphysis, and flushed-out bone marrow and examined mRNA expression. The results showed no significant changes of RANKL and OPG mRNA expression in any part of the femur. In addition, OVX did not significantly affect RANKL and OPG mRNA expression by the adherent stromal cells isolated from flushed-out bone marrow cells but did stimulate RANKL mRNA expression by B220(+) cells in the nonadherent cell fraction. On the other hand, OVX increased IL-7 mRNA expression in the femur as well as IL-7 concentrations in bone fluid. In cultures of unfractionated bone cells isolated by vigorous agitation of minced whole long bones to release the cells tightly attached to the bone surfaces, but not in cocultures of clonal osteoblasts/stromal cells and flushed-out bone marrow cells, IL-7 stimulated generations of osteoclasts as well as osteoclast precursors. These data suggest that increased RANKL production by osteoblasts/stromal cells is unlikely to play a central role in acceleration of osteoclastogenesis in estrogen deficiency of mice and that IL-7 stimulates osteoclast precursor generation, presumably through an action of IL-7 on the cells attached to bone rather than on cells contained in the bone marrow cell population.

Nitrated Fatty Acids: Endogenous Anti-inflammatory Signaling Mediators

Nitroalkene derivatives of linoleic acid (LNO2) and oleic acid (OA-NO2) are present; however, their biological functions remain to be fully defined. Herein, we report that LNO2 and OA-NO2 inhibit lipopolysaccharide-induced secretion of proinflammatory cytokines in macrophages independent of nitric oxide formation, peroxisome proliferator-activated receptor-gamma activation, or induction of heme oxygenase-1 expression. The electrophilic nature of fatty acid nitroalkene derivatives resulted in alkylation of recombinant NF-kappaB p65 protein in vitro and a similar reaction with p65 in intact macrophages. The nitroalkylation of p65 by fatty acid nitroalkene derivatives inhibited DNA binding activity and repressed NF-kappaB-dependent target gene expression. Moreover, nitroalkenes inhibited endothelial tumor necrosis factor-alpha-induced vascular cell adhesion molecule 1 expression and monocyte rolling and adhesion. These observations indicate that nitroalkenes such as LNO2 and OA-NO2, derived from reactions of unsaturated fatty acids and oxides of nitrogen, are a class of endogenous anti-inflammatory mediators.

Tumor Necrosis Factor-α Increases Circulating Osteoclast Precursor Numbers by Promoting Their Proliferation and Differentiation in the Bone Marrow through Up-regulation of c-Fms Expression

Osteoclasts are essential cells for bone erosion in inflammatory arthritis and are derived from cells in the myeloid lineage. Recently, we reported that tumor necrosis factor-alpha (TNFalpha) increases the blood osteoclast precursor (OCP) numbers in arthritic patients and animals, which are reduced by anti-TNF therapy, implying that circulating OCPs may have an important role in the pathogenesis of erosive arthritis. The aim of this study is to investigate the mechanism by which TNFalpha induces this increase in OCP frequency. We found that TNFalpha stimulated cell division and conversion of CD11b+/Gr-1-/lo/c-Fms- to CD11b+/Gr-1-/lo/c-Fms+ cells, which was not blocked by neutralizing macrophage colony-stimulating factor (M-CSF) antibody. Ex vivo analysis of monocytes demonstrated the following: (i) blood CD11b+/Gr-1-/lo but not CD11b-/Gr-1- cells give rise to osteoclasts when they were cultured with receptor activator NF-kappaB ligand and M-CSF; and (ii) TNF-transgenic mice have a significant increase in blood CD11b+/Gr-1-/lo cells and bone marrow proliferating CD11b+/Gr-1-/lo cells. Administration of TNFalpha to wild type mice induced bone marrow CD11b+/Gr-1-/lo cell proliferation, which was associated with an increase in CD11b+/Gr-1-/lo OCPs in the circulation. Thus, TNFalpha directly stimulates bone marrow OCP genesis by enhancing c-Fms expression. This results in progenitor cell proliferation and differentiation in response to M-CSF, leading to an enlargement of the marrow OCP pool. Increased marrow OCPs subsequently egress to the circulation, forming a basis for elevated OCP frequency. Therefore, the first step of TNF-induced osteoclastogenesis is at the level of OCP genesis in the bone marrow, which represents another layer of regulation to control erosive disease.

Stable Isotope Tagging of Epitopes

Identification of peptides presented in major histocompatibility complex (MHC) class I molecules after viral infection is of strategic importance for vaccine development. Until recently, mass spectrometric identification of virus-induced peptides was based on comparative analysis of peptide pools isolated from uninfected and virus-infected cells. Here we report on a powerful strategy aiming at the rapid, unambiguous identification of naturally processed MHC class I-associated peptides, which are induced by viral infection. The methodology, stable isotope tagging of epitopes (SITE), is based on metabolic labeling of endogenously synthesized proteins during infection. This is accomplished by culturing virus-infected cells with stable isotope-labeled amino acids that are expected to be anchor residues (i.e. residues of the peptide that have amino acid side chains that bind into pockets lining the peptide-binding groove of the MHC class I molecule) for the human leukocyte antigen allele of interest. Subsequently these cells are mixed with an equal number of non-infected cells, which are cultured in normal medium. Finally peptides are acid-eluted from immunoprecipitated MHC molecules and subjected to two-dimensional nanoscale LC-MS analysis. Virus-induced peptides are identified through computer-assisted detection of characteristic, binomially distributed ratios of labeled and unlabeled molecules. Using this approach we identified novel measles virus and respiratory syncytial virus epitopes as well as infection-induced self-peptides in several cell types, showing that SITE is a unique and versatile method for unequivocal identification of disease-related MHC class I epitopes.

Immunological evaluation of ex vivo expanded umbilical cord blood T lymphocytes

Objective：The objectives of this study were, first to characterize the immunological differences of the T lymphocytes of umbilical cord blood (UCB) compared to those of adult; second to expand the T lymphocytes in ex vivo condition of a short term culture, and to assess the immunological function of the expanded T lymphocytes. Methods：The immunophenotypic study of 40 UCB and 10 adult peripheral blood (PB) was performed. The fresh UCB mononuclear cells (MNCs) were isolated. The nonadherent MNC fractions were then cultured with the anti-CD3 antibody with or without Loranthus yadoriki (10g/ml). The MNCs were cultured in the anti-CD3 antibody-coated flasks for 4 days, and then transferred to the non-coated flasks, which were added IL-2 175 U/ml and cultured for another 10 days. Proliferative ability of UCB T lymphocytes, cell surface markers, and cytotoxicity assays of the expanded T lymphocytes were performed. Results：Cell surface markers of the UCB T lymphocytes were different from those of adult T cells. The UCB T lymphocytes cultured with the anti-CD3 antibody 100 ng/ml showed a significant increase in the proliferative ability (p＜0.05). After culture in the anti-CD3 antibody coated flask with IL-2, expression of the activated T lymphocytes were increased significantly. The cultured cells exhibited substantial killing activity on the SK-OV-3 target cells compared to the fresh UCB lymphocytes. Conclusion：The ex vivo combination of the anti-CD3 antibody and IL-2 significantly enhanced proliferation, activation, and maturation of the UCB T lymphocytes. Moreover cytotoxic potential of expanded UCB T cells was observed.

Functional Comparison of Bone Marrow–Derived Liver Stem Cells: Selection Strategy for Cell-Based Therapy

Several distinct subpopulations of bone marrow-derived liver progenitor cells were recently described. However, there is inadequate information comparing these subpopulations from a liver-function point of view. This study was undertaken to compare two subpopulations of liver progenitors: beta(2)-microglobulin (beta(2)m)-negative/Thy-1-positive cells, and liver progenitors obtained from the non-adherent cell fraction after a panning procedure. The cells were cultured under several conditions including high- and low-dose hepatocyte growth factor, various cellular densities, and different media. Growth characteristics, liver-specific metabolic capacity, and liver regeneration-associated gene expression were studied. Both isolation procedures yielded cells that produced albumin and metabolized ammonia into urea. The study demonstrated that the beta(2)m-negative/Thy-1-positive cell fraction metabolized ammonia into urea more efficiently and produced a superior amount of albumin compared with the panned cell fraction. The beta(2)m-negative/Thy-1-positive cell fraction could be optimal for the development of novel cell-based treatment strategies for congenital or acquired liver diseases.

Induction of CML28-Specific Cytotoxic T Cells by Dendritic Cells Cotransfected with CML28 Nucleic Acid Vaccination and SOCS1-Specific siRNA Expression Vector In Vitro.

CML28 is a new tumor-associated antigen overexpressed in tumor cells, which is an attractive target for antigen-specific immunotherapy. In this study, we evaluated the possibility to induce CML28-specific cytotoxic T cells by cotransfection of CML28 nucleic acid vaccination and SOCS1-specific siRNA expression vector with dendritic cells in vitro. The full length CML28 cDNA was amplified from K562 by RT-PCR and cloned into a bicistronic vector pIRES2-EGFP to construct the CML28 nucleic acid vaccination. Dendritic cells (DCs) were generated from peripheral blood mononuclear cells (PBMNCs) of HLA-A2+ healthy donor. Construct the recombinant plasmid psiRNA-hH1neo-SOCS1 encoding hairpin small interfering RNA (siRNA) targeting to suppressors of cytokine signaling 1 (SOCS1) sequences using a vector-base RNA interference technology. Cotransfect CML28 nucleic acid vaccination and recombinant siRNA vector psiRNA-hH1neo-SOCS1 into DCs by electroporation. Real-time RT-PCR was performed to validate the SCOS1 gene silencing efficacy. The expression products of CML28 nucleic acid vaccination, His-CML28 fusion protein and GFP protein, were measured by Western Blotting and fluorescence microscope respectively. Labeling autologous nonadherent fraction of PBMNCs as responder cells by 5, 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) firstly, and then coculture with cotransfected DCs for mixed lymphocyte reaction (MLR). Autogous T cell proliferative response of MLR was measured by FACS by detecting CFSE. Cotransfected DCs and the PBMNCs of a HLA-A2+ primary leukemia patient were used as target cells, untransfected DCs, K562 and HL60 were used as control. The standard 51Cr-release assay was performed to measure cytotoxicity of stimulated lyphocytes. CML28 nucleic acid vaccination could encode His-CML28 and GFP protein in DCs successfully. SOCS1-specific siRNA expression vector psiRNA-hH1neo-SOCS1 significantly suppress the expression of SOCS1 in DCs. Down regulation of SOCS1 resulted in higher expression level of CD80, CD86 and CD83 in cotranfected DCs and more rounds of cell division of responder cells in CFSE-MLR, which indicates SOCS1 gene silencing greatly contribute to maturation of DCs and enhance the the proliferative response of responder cells. CTLs induced by cotransfected DCs exhibit stronger CML28-specific cytotoxicity against HLA- matched target cells comparing to CTLs induced by CML28 nucleic acid vaccination transfected DCs only. SOCS1 gene silencing in DCs could greatly enhance the anti-tumor efficacy of CML28 nucleic acid vaccination. DCs cotransfected with CML28 nucleic acid vaccination and SOCS1-specific siRNA expression vector could effectively induce autologous CML28-specific cytotoxic T cells that lyse CML28 positive tumor cells in an antigen-specific and major histocompatibility complex (MHC) class I-restricted manner.

Normal bone marrow hematopoietic stem cell reserves and normal stromal cell function support the use of autologous stem cell transplantation in patients with multiple sclerosis

Bone marrow (BM) stem cell reserves and function and stromal cell hematopoiesis supporting capacity were evaluated in 15 patients with multiple sclerosis (MS) and 61 normal controls using flow cytometry, clonogenic assays, long-term BM cultures (LTBMCs) and enzyme-linked immunosorbent assays. MS patients displayed normal CD34+ cell numbers but a low frequency of colony-forming cells (CFCs) in both BM mononuclear and purified CD34+ cell fractions, compared to controls. Patients had increased proportions of activated BM CD3+/HLA-DR+ and CD3+/CD38+ T cells that correlated inversely with CFC numbers. Patient BM CD3+ T cells inhibited colony formation by normal CD34+ cells and patient CFC numbers increased significantly following immunomagnetic removal of T cells from BMMCs, suggesting that activated T cells may be involved in the defective clonogenic potential of hematopoietic progenitors. Patient BM stromal cells displayed normal hematopoiesis supporting capacity indicated by the CFC number in the nonadherent cell fraction of LTBMCs recharged with normal CD34+ cells. Culture supernatants displayed normal stromal derived factor-1 and stem cell factor/kit ligand but increased flt-3 ligand levels. These findings provide support for the use of autologous stem cell transplantation in MS patients. The low clonogenic potential of BM hematopoietic progenitors probably reflects the presence of activated T cells rather than an intrinsic defect.

Osteopetrotic Mouse Stroma with Thrombopoietin, c-kit Ligand, and flk-2 Ligand Supports Long-Term Mobilized CD34+Hematopoiesis In Vitro

OP-9 cells are stromal cells derived from macrophage colony-stimulating factor (M-CSF)-deficient osteopetrotic mice. To evaluate the OP-9 capability to sustain long-term hematopoiesis, we reported the expansion of granulocyte colony-stimulating factor (G-CSF)-mobilized human peripheral blood (PB) CD34(+) cells in co-culture with murine OP-9 and MS-5 stromal cells, either transfected with various combinations of adenovectors (Ad) expressing c-kit ligand (KL) (either soluble or transmembrane form), thrombopoietin (TPO), flt-3/flk2 ligand (FL), and granulocyte-macrophage (GM)-CSF or with weekly addition of these cytokines. Expression of TPO as well as association of TPO, FL, and KL increased progenitor cell and week-6 cobblestone area forming cell (CAFC) production in all stromal co-cultures. Similar progenitor expansion was obtained by weekly addition of soluble cytokine. Five weeks of co-culture with OP9 and TPO, FL + KL resulted in the greatest expansion of progenitor cells and week-6 CAFC as measured by secondary assay on MS-5. In contrast to MS-5 and TPO or TPO + FL + KL cultures where hematopoiesis declined by week 4, progenitor as well as week-6 CAFC expansion continued for over 3 months in TPO + FL + KL OP9 cocultures. This was associated with decrease of CD14(+) macrophage production. The addition of human macrophage (M)-CSF or CD14(+) cells to the co-culture decrease progenitor and stem cell expansion; however, murine M-CSF to OP-9 co-cultures did not decrease progenitor expansion. High levels of stromal-derived factor-1 (SDF-1) production by MS-5 and low or absent production by OP-9 may account for stem cell adhesion and CAFC formation in the former cultures and the predominance of stem and progenitor cells in the nonadherent fraction in the latter cultures.

Regulation of CTL responses to MHC‐restricted class I peptide of the gp70 tumour antigen by splenic parenchymal CD4+ T cells in mice failing immunotherapy with DISC–mGM‐CSF

Direct intratumour injection of the disabled infectious single-cycle-herpes simplex virus-encoding murine granulocyte/macrophage colony-stimulating factor (DISC-HSV-mGM-CSF) into established colon carcinoma CT26 tumours induced complete tumour rejection in up to 70% of treated animals (regressors), while the remaining mice developed progressive tumours (progressors). This murine Balb/c model was used to dissect the cellular mechanisms involved in tumour regression or progression following immunotherapy. CTLs were generated by coculturing lymphocytes and parenchymal cells from the same spleens of individual regressor or progressor animals in the presence of the relevant AH-1 peptide derived from the gp70 tumour-associated antigens expressed by CT26 tumours. Tumour regression was correlated with potent CTL responses, spleen weight and cytokine (IFN-gamma) production. Conversely, progressor splenocytes exhibited weak to no CTL activity and poor IFN-gamma production, concomitant with the presence of a suppressor cell population in the progressor splenic parenchymal cell fraction. Further fractionation of this parenchymal subpopulation demonstrated that cells inhibitory to the activation of AH-1-specific CTLs, restimulated in vitro with peptide, were present in the nonadherent parenchymal fraction. In vitro depletion of progressor parenchymal CD3+/CD4+ T cells restored the CTL response of the cocultured splenocytes (regressor lymphocytes and progressor parenchymal cells) and decreased the production of IL-10, suggesting that CD3+CD4+ T lymphocytes present in the parenchymal fraction regulated the CTL response to AH-1. We examined the cellular responses associated with tumour rejection and progression, identifying regulatory pathways associated with failure to respond to immunotherapy.

Ex Vivo Expansion of Human Umbilical Cord Blood-Derived T-Lymphocytes with Homologous Cord Blood Plasma

This study was designed to establish a more effective and safe culture system for adoptive immunotherapy by investigating the use of homologous cord blood plasma (HCBP) instead of fetal bovine serum (FBS), which has various limitations including ethical problems for the ex vivo expansion of human umbilical T lymphocytes. Fresh human umbilical mononuclear cell fractions were isolated by Ficoll-Hypaque density centrifugation. Nonadherent mononuclear cell fractions were cultured with anti-CD3 antibody (5 microg/ml), IL-2 (175 U/ml), and either 10% FBS or 10% HCBP. On day 8, the cellular proliferation rate and cell surface markers were assessed. There was no significant difference in proliferation when human umbilical cord blood T lymphocytes were grown in medium supplemented with FBS or HCBP (p > 0.05). In medium containing FBS, the proportion of CD3(+)CD4(+) (markers for helper T cell), CD3(+)CD8(+) (cytotoxic T cell), CD3(+)CD25(+) (activated T cell), CD3(+)CD38(+) (immature T cell), and CD3(+)CD45RO(+) (memory T cell) cells was significantly increased (p < 0.05), whereas proportion of CD3(+)CD45RA(+) (naive T cell) and CD16(+)CD56(+) (NK cell) cells was significantly decreased (p < 0.05). In HCBP supplemented medium, the proportion of CD3(+)CD8(+), CD3(+)CD25(+), CD3(+)CD45RA(+), and CD3(+)CD45RO(+) cells was significantly increased (p < 0.05). The proportion of CD3(+)CD4(+), CD3(+)CD45RO(+) and CD3(+)CD38(+) cells was significantly higher, but proportion of CD3(+)CD45RA(+) and CD3(+)CD8(+) cells was significantly lower in FBS compared with HCBP supplemented medium (p < 0.05). Our results support the feasibility of ex vivo expansion of human umbilical cord blood T lymphocytes in medium supplemented with HCBP for future adoptive cellular immunotherapy.

The Addition of Interleukin (IL)-15 to ANTI-CD3, IL-2 and FLT-3 Results in Significant Increase in Expansion, Activation, Proliferation and Survival of Cord Blood (CB) NK Cells and Cytotoxic T-Lymphocyte (CTL) Subsets: Potential for Adoptive Cellular Immunotherapy (ACI).

A limitation of CB as a source for ACI is the limited number of immunoeffector T and NK cell subsets. IL-15 is an important lymphokine regulating NK activity, T-cell proliferation and CTL activities. We have previously demonstrated reduced IL-15 mRNA expression and protein production from CB which may in part contribute to the immaturity of CB cellular immunity (Qian/Cairo et al, Blood 1997). NK cells are characterized by NK receptors which exist in both activatory and inhibitory forms (Moretta et al, Ann Rev Immun 2001). We have previously demonstrated the ex vivo expansion, maturation and activation of fresh CB derived T lymphocytes with anti-CD3, IL-2, IL-12 and IL-7 (Robinson/Cairo et al, Exp Hem 2001). For this study, we compared the expansion, proliferation, activation and survival of CB mononuclear cells (MNC) with IL-2, anti-CD3, FLT-3 ± IL-15. CB MNC were isolated by Ficoll density centrifugation and incubated overnight in AIM-V media. The non-adherent MNC fraction was cultured in AIM-V media alone and with IL-2 (5ng/ml), anti-CD3 (50ng/ml), FLT-3 (50ng/ml) and IL-15 (10 &100ng/ml) for 48 hrs. Cell proliferation was determined by the colorimetric assay system using WST-1. Expression of T & NK cell subsets were analyzed by three color flow cytometry using CD3, CD8, CD4, CD25, CD45RO, CD16, CD56, CD158a, CD158b, NKB1, CD94 & NKG2A monoclonal antibodies. Apoptosis markers were determined by measuring Annexin-V & PI by flow cytometry. Cytotoxicity was determined by europium release assay using K562 & Daudi target cells at 20:1 E:T ratio. Expansion of lymphocyte subsets of CD4+/25+, CD8+/25+, CD3+/45RO+ & CD94/NKG2A was significantly increased with IL-2, anti-CD3, FLT-3 + IL-15(100ng/ml) compared to IL-2, anti-CD3, FLT-3 and compared to media (CD4+/25+: 67.2±1.2 vs 39.19±5.3 vs 3.54±0.3%, p<0.01; CD8+/25+: 57.8±8.6 vs 16.94±0.3 vs 0.99±0.4%, p<0.01; CD3+/45RO+: 77.13±1.0 vs 29.96±1.9 vs 6.69±0.3%, p<0.001; CD94/NKG2A: 3.4±0.68 vs 0.29±0.092 vs 0.23±0.09%, p<0.01, respectively). We also noted a significant increase in CD3−/16+/56+ with IL-2, anti-CD3, FLT-3 + IL-15(100ng/ml) compared to media alone (p<0.01). There was no significant increase in expansion of CD3+/16+/56+, CD3−/56+/158a, CD3−/56+/158b or CD3−/56+/NKB1 in various culture conditions described above. IL-2, anti-CD3, FLT-3 + IL-15(100ng/ml) resulted in significant improvement in cell survival compared to media alone (Annexin-V staining) (3.3±0.26 vs 15.51±3.3%, p<0.05). We also noted increase in cell proliferation with IL-2, anti-CD3, FLT-3 + IL-15(100ng/ml) as compared to IL-2, anti-CD3, FLT-3 vs. media alone (1.11±0.09 vs 0.82±0.01 vs 0.69±0.03 OD, p<0.05). We also noted significant increase in cytotoxicity with IL-2, anti-CD3, FLT-3 + IL-15(100ng/ml) as compared to IL-2, anti-CD3, FLT-3 vs media alone (K562–57.81±8.1 vs 17.5±1.4 vs 2.68±0.6%, p<0.01; Daudi-79.8±1.7 vs 23.69±1.5 vs 6.38±0.6, p<0.001) The combination of IL-2 + anti-CD3 + FLT-3 + IL-15 appears to enhance expansion and cytotoxicity of CB CTL and NK subsets in short term culture in part secondary to inhibition of apoptosis. Future studies are warranted to determine if the combination of anti-CD3 + IL-2 + FLT-3 + IL-15 can enhance CB CTL and NK cells for ACI to induce GVHM effects following UCBT.