Immunoselected Cells Research Articles

Randomized Trial of Targeted Transendocardial Mesenchymal Precursor Cell Therapy in Patients With Heart Failure

BackgroundMesenchymal precursor cells (MPCs) are allogeneic, immunoselected cells with anti-inflammatory properties that could improve outcomes in heart failure with reduced ejection fraction (HFrEF). ObjectivesThis study assessed the efficacy and safety of MPCs in patients with high-risk HFrEF. MethodsThis randomized, double-blind, multicenter study evaluated a single transendocardial administration procedure of MPCs or sham-control in 565 intention-to-treat patients with HFrEF on guideline-directed therapies. The primary endpoint was time-to-recurrent events caused by decompensated HFrEF or successfully resuscitated symptomatic ventricular arrhythmias. Hierarchical secondary endpoints included components of the primary endpoint, time-to-first terminal cardiac events, and all-cause death. Separate and composite major adverse cardiovascular events analyses were performed for myocardial infarction or stroke or cardiovascular death. Baseline and 12-month echocardiography was performed. Baseline plasma high-sensitivity C-reactive protein levels were evaluated for disease severity. ResultsThe primary endpoint was similar between treatment groups (HR: 1.17; 95% CI: 0.81-1.69; P = 0.41) as were terminal cardiac events and secondary endpoints. Compared with control subjects, MPCs increased left ventricular ejection fraction from baseline to 12 months, especially in patients with inflammation. MPCs decreased the risk of myocardial infarction or stroke by 58% (HR: 0.42; 95% CI: 0.23-0.76) and the risk of 3-point major adverse cardiovascular events by 28% (HR: 0.72; 95% CI: 0.51-1.03) in the analysis population (n = 537), and by 75% (HR: 0.25; 95% CI: 0.09-0.66) and 38% (HR: 0.62; 95% CI: 0.39-1.00), respectively, in patients with inflammation (baseline high-sensitivity C-reactive protein ≥2 mg/L). ConclusionsThe primary and secondary endpoints of the trial were negative. Positive signals in prespecified, and post hoc exploratory analyses suggest MPCs may improve outcomes, especially in patients with inflammation.

Infrapatellar fat pad-derived mesenchymal stem cell-based spheroids enhance their therapeutic efficacy to reverse synovitis and fat pad fibrosis

BackgroundTo investigate the in vitro and in vivo anti-inflammatory/anti-fibrotic capacity of IFP-MSC manufactured as 3D spheroids. Our hypothesis is that IFP-MSC do not require prior cell priming to acquire a robust immunomodulatory phenotype in vitro in order to efficiently reverse synovitis and IFP fibrosis, and secondarily delay articular cartilage damage in vivo.MethodsHuman IFP-MSC immunophenotype, tripotentiality, and transcriptional profiles were assessed in 3D settings. Multiplex secretomes were assessed in IFP-MSC spheroids [Crude (non-immunoselected), CD146+ or CD146− immunoselected cells] and compared with 2D cultures with and without prior inflammatory/fibrotic cell priming. Functionally, IFP-MSC spheroids were assessed for their immunopotency on human PBMC proliferation and their effect on stimulated synoviocytes with inflammation and fibrotic cues. The anti-inflammatory and anti-fibrotic spheroid properties were further evaluated in vivo in a rat model of acute synovitis/fat pad fibrosis.ResultsSpheroids enhanced IFP-MSC phenotypic, transcriptional, and secretory immunomodulatory profiles compared to 2D cultures. Further, CD146+ IFP-MSC spheroids showed enhanced secretory and transcriptional profiles; however, these attributes were not reflected in a superior capacity to suppress activated PBMC. This suggests that 3D culturing settings are sufficient to induce an enhanced immunomodulatory phenotype in both Crude and CD146-immunoselected IFP-MSC. Crude IFP-MSC spheroids modulated the molecular response of synoviocytes previously exposed to inflammatory cues. Therapeutically, IFP-MSC spheroids retained substance P degradation potential in vivo, while effectively inducing resolution of inflammation/fibrosis of the synovium and fat pad. Furthermore, their presence resulted in arrest of articular cartilage degradation in a rat model of progressive synovitis and fat pad fibrosis.Conclusions3D spheroids confer IFP-MSC a reproducible and enhanced immunomodulatory effect in vitro and in vivo, circumventing the requirement of non-compliant cell priming or selection before administration and thereby streamlining cell products manufacturing protocols.

The Challenge of Using CB-HSCs As Source for Gene Therapy: Lentiviral Vector Transduction, Phenotypic Characterization and Global Gene Expression Profile of Ex-Vivo Expanded CB CD34+ Cells

Introduction: Genetic modification of autologous hematopoietic stem and progenitor cells (HSPC) is a promising clinical intervention to cure inherited monogenic diseases. Successful gene therapy trials have already been conducted using CD34+ cells from bone marrow and from mobilized peripheral blood. In this regard, cord blood (CB) represents an attractive source of HSCs due to its high concentration of high proliferative HSPC and increased susceptibility to be transduced by lentiviral vectors. Unfortunately, the major disadvantage is the limited number of HSC in the CB collection. Consequently, ex-vivo expansion of CB-HSC is desirable to extend clinical applications.Purposes: To investigate the ability of UCB-cd34+ cells to be expanded in serum-free media supplemented with the early acting hematopoietic cytokines SCF,TPO and Flt-3 ligant (STF) and to characterize CD34+ cells subtypes, clonogenic capacity and gene expression profile during expansion. We also wanted to investigate the susceptibility of the expanded cd34+ cells to be transduced by a GFP-lentiviral vector (LV-GFP)Material and Methods: CD34+ immunoselected cells from 10 UCB were grown for 8 days in customized serum-free medium formulated for HSC expansion, supplemented with STF cytokines. Numbers end frequency of CD34+cells and co-expression of the primitive surface antigens (CD38, CD133, CD90) was evaluated during expansion. Colonies developed in methylcellulose were scored for enumeration ad typing.LV-GFP transduction efficiency was evaluated in CD34+ cells cultured for 4 days in expansion medium plus STF and for 24 hrs in X-vivo10 medium with STF±IL-3 cytokines; the last condition slightly expands CD34+ cells (1.3 fold) and are currently used for HSPC-lentivector transduction in gene therapy clinical trials.The transduction efficiency was evaluated by measuring the percentage of GFP+ cells in the bulk and in colonies developed in methylcellulose and the VCN/cell by Q-PCR.Gene expression profiles were analyzed by human whole genome Agilent microarray Technology to detect differentially expressed genes between expanded, ex-vivo medium cultured and un-cultured cells.Results: We found an average of 8 fold-increase CD34+cells at day 4 and of 22 fold- increase at day 8 of culture. The frequency of CD34+ was maintained at day 4 and declined of about 50% at day 8. CD34+/CD38- early progenitors doublet as early as day 4, differences in CD34+/CD133+ and CD34+/CD90+cells were not significant.The number of CFU slightly increased during expansion while the relative frequency of colonies type did not significantly changed.Four days expanded CD34+ cells were transduced more efficiently than those grown in ex-vivo medium even in presence of IL-3 added to the STF cytokine cocktail.Comprehensive gene expression profile analysis highlighted about 4000 genes differentially expressed in CD34+ cells expanded for 4 and for 8 days compared to that of the un-cultured cells. Conversely, the expression profiles analysis did not show any clear separation between different cell culture methods (expansion vs ex-vivo medium). Specifically, the number of differentially expressed genes in common between the different culture conditions compared with the un-cultured cells was statistically significant. Unsurprisingly, the common up-regulated genes were related to the cell cycle. The likeness between the gene expression profiles of the different culture conditions was also validated by the identification of a significantly small number of differentially expressed genes between them.Conclusions: UCB-CD34+ cells can be efficiently expanded and transduced in serum free conditions. The expanded cells exhibited phenotypic marchers typical of early progenitors and developed colonies in number and in type similar to the unmanipulated cells and exhibited whole gene expression profile that is consisted with that of CD34+ cells exposed for the short term culture conditions currently used in gene therapy trial mediated by lentiviral vectors.Results from this study open a window on the future possibility of using homologous UCB-HSC as target for gene correction in patients diagnosed for a genetic disorder in prenatal time.The genetically modified cells would be stored and used for gene therapy in the same individual in pediatric age.This work was funded by the F and P Cutino Foundation - Project RiMedRi CUP G73F12000150004 DisclosuresNo relevant conflicts of interest to declare.

Interaction between human mature adipocytes and lymphocytes induces T-cell proliferation

Background aimsAdipose tissue is a critical organ that plays a major role in energy balance regulation and the immune response through intricate signals. MethodsWe report on the inter-relation between mature adipocytes and lymphocytes in terms of adipocyte-derived T-cell chemo-attractants and adipocyte metabolic effects on lymphocytes. ResultsDuring the culture time, mature adipocytes changed their structural and functional properties into de-differentiated cells. Isolated mature adipocytes expressed significantly higher levels of CIITA, major histocompatibility complex II (human leukocyte antigen [HLA]-DR) and costimulatory signal molecule CD80 compared with adipocytes after the de-differentiation process. Moreover, human leukocyte antigen–G, which may prevent the immune responses of mesenchymal stromal cells, was expressed at lower level in mature adipocytes compared with de-differentiated adipocytes. In line with these molecular data, functional results showed different immunoregulatory properties between adipocytes before and after the de-differentiation process. Mature adipocytes stimulated the proliferation of total lymphocytes and immunoselected cell populations CD3+, CD4+ and CD8+ in a direct contact-dependent way that involved the major histocompatibility complex I and II pathways. Moreover, adipocytes secreted potential chemo-attractant factors, but data showed that adipocyte-derived culture medium was not sufficient to activate lymphocyte proliferation, suggesting that a direct contact between adipocytes and immune cells was needed. However, specific mature adipocyte cytokines enhanced lymphocyte proliferation in a mixed lymphocyte reaction. ConclusionsIn conclusion, cross-talk occurs between adipocytes and lymphocytes within adipose tissue involving T-cell chemo-attraction by mature adipocytes. Our findings, together with current observations in the field, provide a rationale to identify adipocyte-lymphocyte cross-talk that instigates adipose inflammation.

Abstract 5226: In vivo imaging and molecular characterization of site-specific growth of malignant melanoma: a study of melanoma metastasis in experimental animal models

Abstract Metastatic melanoma is a very aggressive and fatal disease in need of novel therapeutic targets. Melanomas are notorious for the ability to disseminate to almost any organ. The ability to develop metastases at distant sites is not only a function of intrinsic properties of the malignant cells themselves, but is also influenced by a wide range of factors in the host microenvironment. Understanding of the biological role of the microenvironment in the establishment of metastases and identifying implicated factors, might uncover novel potential targets for anti-metastatic therapy. The aim of our study was to reveal characteristics of metastatic growth of melanoma in different anatomical sites in animal models through in vivo imaging and molecular approaches. We have developed experimental metastasis models in nude mice based on intracardial (left ventricle) injection of the melanoma cell lines Melmet 1 and Melmet 5, established from two metastatic melanoma patients with different clinical presentations. A follow-up of melanoma cell migration and micro/macrometastases formation assessed by molecular and functional imaging in vivo (IVIS and PET), indicated that Melmet 5 could establish metastases in multiple organs (including brain, lung, lymph nodes, kidney, bone), while Melmet 1 displayed a more consistent preference for the brain. Flow cytometric analysis of metastatic organs, specifically brain and lungs, revealed the recruitment of bone marrow derived cells, identified as CD11b+/Gr-1+ myeloid cells and CD11b+/F4/80+ macrophages. These cell subpopulations have been linked to the formation of a (pre)metastatic niche, and we are presently investigating their contribution to the metastatic growth of melanoma. Further, we performed a comparative microarray study to investigate differential gene expression with respect to organ-specific environments. Specifically, we identified a set of genes altered in immunoselected melanoma cells purified from brain tissue. Among these genes were receptors involved in glutamate signaling, indicating metabolic changes associated with metastatic colonization of the brain. Furthermore, after separation of tumor cells, the remaining host brain tissue was also analyzed by gene expression profiling in order to reveal molecular changes induced in the stroma compartment by metastases. Compared to non-diseased brain, we found altered expression of genes implicated in biological activities such as immune defense, evasion of apoptosis and cell adhesion and migration. Based on the results obtained, we are currently performing functional studies on candidate genes to elucidate their role in metastatic growth of melanoma. Targeting focal points of communication between metastatic tumor cells and the host in distinct microenvironments may represent an anti-metastatic therapeutic strategy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5226. doi:10.1158/1538-7445.AM2011-5226

Gene expression profile of cytokines in patients with chronic graft-versus-host disease after allogeneic hematopoietic stem cell transplantation with reduced conditioning

There are no reliable markers useful to predict the onset or the evolution of chronic graft-versus-host disease (cGVHD) after allogeneic hematopoietic stem cell transplantation (HSCT), although several candidate biomarkers have been identified from limited hypothesis-driven studies. In this study we evaluated 14 patients who received a reduced intensity conditioning HSCT. Seven patients had cGVHD, whereas 7 never developed cGVHD during the period of observation. The expression of 114 cytokines in immunoselected cell populations was explored by microarray analysis and 11 cytokines were selected for further evaluation by real-time PCR. Differential gene expression measurements showed a significant up-regulation for INFγ (interferon, gamma) in CD8+ and for TNFSF3 (tumor necrosis factor superfamily, member 3) and for TNFSF10 (tumor necrosis factor superfamily, member 10) in CD14+ cell population when comparing cGVHD with control samples. The expression levels were significantly decreased for TNFSF10 in CD8+ cell population and for TNFSF12 (tumor necrosis factor superfamily, member 12) and for PDGFβ (platelet-derived growth factor, beta) in CD4+. Our data seem to suggest that different immune populations can play a role in cGVHD pathogenesis and the early detection of gene expression profile in these patients could be useful in the monitoring of GVHD. We hypothesized that PDGFβ down-regulation could represent a negative feedback to compensate for enhanced expression of its receptor recently reported.

Abstract PD02-05: Low Immunogenicity of Breast Cancer Stem Cells Leads to Selective Survival When Challenged with Trastuzumab and Natural Killer Cells

Abstract Although trastuzumab (Herceptin) has substantially improved the overall survival of patients with mammary carcinomas, even initially well-responding tumors often become resistant. Considering that clinical efficacy of trastuzumab correlates with the infiltration of natural killer (NK) cells into the tumor site, we established a cell-culture-system to select for ovarian cancer and mammary carcinoma cells that survive a challenge by trastuzumab and NK cells. Under these conditions, NK cells can eliminate tumor cells either via antibody-dependent-cell-mediatedcytotoxicity (ADCC) or by direct recognition of tumor-associated ligands for NKG2D or DNAM-1. The most striking phenotypic alteration observed in immunoselected ovarian cancer cells was a down-regulation of HER2 expression, leading to an ADCC-resistant phenotype. All breast cancer cells tested (MCF7, SK-BR-3, MDA-MB231, BT474), however, failed to develop resistance in vitro. Instead, treatment with trastuzumab and polyclonal NK-cells resulted in the preferential survival of individual sphere-forming cells that displayed a CD44highCD24low ‘'cancer-stem-celllike” phenotype (CSC) and expressed significantly less HER2 compared with non-stem cells. Moreover, the molecular determinants for the direct recognition of transformed cells, most notably the NKG2D ligands MICA, MICB, ULBP1-4 and the DNAM-1 ligands CD112 and CD155, were virtually absent from CSC. When immunoseleced breast cancer cells were then re-expanded, they mostly lost the observed phenotype and regenerated a tumor cell culture that displayed initial HER2 surface expression and ADCC susceptibility, but was enriched in CD44highCD24low CSC. This translated into increased clonogenicity in vitro and tumorigenicity in vivo. We provide evidence that recruition of NK-cells and induction of ADCC by trastuzumab may spare actual tumor-initiating-cells, which could explain clinical relapse. Moreover, our observation that “relapsed” in vitro cultures show identical HER2 surface expression and susceptibility toward ADCC suggests that administration of trastuzumab beyond relapse might be considered, especially when combined with proteasome inhibition which increase the expression of NKG2D ligands. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD02-05.

Paracrine signals from mesenchymal cell populations govern the expansion and differentiation of human hepatic stem cells to adult liver fates.

The differentiation of embryonic or determined stem cell populations into adult liver fates under known conditions yields cells with some adult-specific genes but not others, aberrant regulation of one or more genes, and variations in the results from experiment to experiment. We tested the hypothesis that sets of signals produced by freshly isolated, lineage-dependent mesenchymal cell populations would yield greater efficiency and reproducibility in driving the differentiation of human hepatic stem cells (hHpSCs) into adult liver fates. The subpopulations of liver-derived mesenchymal cells, purified by immunoselection technologies, included (1) angioblasts, (2) mature endothelia, (3) hepatic stellate cell precursors, (4) mature stellate cells (pericytes), and (5) myofibroblasts. Freshly immunoselected cells of each of these subpopulations were established in primary cultures under wholly defined (serum-free) conditions that we developed for short-term cultures and were used as feeders with hHpSCs. Feeders of angioblasts yielded self-replication, stellate cell precursors caused lineage restriction to hepatoblasts, mature endothelia produced differentiation into hepatocytes, and mature stellate cells and/or myofibroblasts resulted in differentiation into cholangiocytes. Paracrine signals produced by the different feeders were identified by biochemical, immunohistochemical, and quantitative reverse-transcription polymerase chain reaction analyses, and then those signals were used to replace the feeders in monolayer and three-dimensional cultures to elicit the desired biological responses from hHpSCs. The defined paracrine signals were proved to be able to yield reproducible responses from hHpSCs and to permit differentiation into fully mature and functional parenchymal cells. Paracrine signals from defined mesenchymal cell populations are important for the regulation of stem cell populations into specific adult fates; this finding is important for basic and clinical research as well as industrial investigations.

An Improved Bicistronic CD20/tCD34 Vector for Efficient Purification and In Vivo Depletion of Gene-Modified T Cells for Adoptive Immunotherapy

T-cell-based adoptive immunotherapy is widely used to treat graft rejection and relapse after stem cell transplantation (SCT). However, this approach is hampered by a high risk of life-threatening graft-versus-host-disease (GvHD). Clinical trials have demonstrated the value of suicide genes to modify T cells for the effective control of GvHD. Herewith, we show that the combination of a codon-optimized B-cell antigen (CD20op) with a selection marker based on a cytoplasmic truncated version of the human stem cell antigen CD34 (tCD34) allows the generation of highly enriched gene-modified T cells. We demonstrate coordinate co-expression of both transgenes and high expression of CD20op resulting in an increased susceptibility to Rituximab (RTX)-induced cell death. In addition, T cells partially retained their alloreactive potential and their CD4/CD8 ratio after transduction and expansion. Long-lasting transgene expression was sustained in vivo after adoptive transfer into Rag-1(-/-) mice. Moreover, gene-modified T cells were quickly and efficiently depleted from peripheral blood (PB) and secondary lymphoid organs of transplanted animals after RTX treatment. These results warrant further steps toward a clinical application of CD20op as a suicide gene for adoptive immunotherapy.

Prominin-1 Mobilisation, Collection and Immunoselection in Cancer Patients for Liver Regeneration.

Abstract 2141Poster Board II-118 Background:CD133+ (Prominin-1 positive) is a 5-transmembrane glycoprotein that identifies immature progenitor stem cells.Immature hematopoietic stem cells retain the possibility to give origin to tissues different from hematopoietic cell lines (transdifferentiation). Material and methods:In this preliminary study we investigated the possibility to mobilize, collect, immunoselect and reinfuse autologous CD133+ immature stem cells in liver cancer patients. This approach was adopted to obtain, in a short time, an adequate volume increase of the disease free liver thus extending the resectability criteria of the liver, with the final goal to prolong survival.We enrolled 4 patients with a large liver cancer and no chance of resection. The mobilization protocol consisted in: G-CSF administration (10 μg/kg/day ) for 3-5 days, peripheral blood stem cell (PBSC) monitoring starting from the 3rd day, leukapheresis (LKF) collection processing 3 blood volumes when CD133+ cells>15 μL. Patients were monitored during mobilization, collection and post collection phase for clinical status, blood pressure and bleeding. Positive CD133+ immunoselection (Miltenyi Clinimacs) was performed on LKF product after overnight storage. Quality controls on positive fraction consisted in viability and purity of CD133+ cells by cytofluorimetric analysis and clonogenic assays. Microbial tests were performed on the negative fraction.After LKF, patients underwent right portal embolization and infusion of CD133+ cells into the opposite portal vein by a percutaneous access. Evaluation of liver regeneration was performed 30 days after stem cell infusion by spiral CT and galactose clearance. Liver resection was carried out if liver regeneration reached 30-40%. Results:Stem cell mobilization, LKF content and immunoselected cells are detailed in tab 1. No relevant side effects were observed. We obtained an efficient stem cell mobilization in all patients enrolled. No bacterial or fungal contamination was observed in cells infused. Results about liver regeneration and patients' follow up are detailed in table 2.Table 1Mobilization, leukapheresis content and characteristics of cells infused.Median pts' weight (range): 79.7 kg (63 -120) Median days for mobilization (range) 4 (3-4) Median WBC count at the time of collection (range) 37.3×106/ml (26-48) Median PBSC at the time of collection (range) CD133+: 21/mL (16-47.7)>CD34+: 41.5/ mL (22-82)Leukapheresis content mean (range) WBC: 113.2×109 (20.4-200.3) CD133+: 172.7×106 (40.1-372.5) CD34+: 265.3×106 (60-592.6)Characteristics of cells infused mean (range) CD133+: 65.7×106 (41-83) CD133+/kg 0.85×106 (0.29-1.35) CD133+ recovery: 38% (26-55) Viability % (7AAD): 98% (96-99) Purity: 94% (91-95)Table 2Liver regeneration after portal vein infusion of CD133+ cells. FLRV(Future Liver Remnant Volume) expressed as volume (cc) and percentage of total liver mass.Case 1Case 2Case 3Case 4Total volume (cc)1745168913082816Basal FLRV (%)441 (25)611/36290/22723/2630 Days post-treatment FLRV (%)920 (48)772/45568/401080/38TumorCholangiocarcinomaHCC multifocal, PV invasionMetastases of rectal cancerMetastases of colon cancerSurgeryResectedProgression of diseaseResectedResectedStatusAlive free from disease (15 months follow-up)Dead (8 months from diagnosis)Alive, residual disease (12 months follow-up )Alive (6 months follow -up), disease progression Conclusions:Our approach to liver regeneration was feasible and safe with no relevant side effects. We observed an efficient stem cell mobilization comparable to healthy donors also in liver cancer patients. The infusion of CD133+ cells allowed a significant hepatic tissue regeneration in all patients. Controlled clinical trials are needed to confirm our preliminary results. Disclosures:No relevant conflicts of interest to declare.

In Vivo Imaging to Monitor Trafficking and Engraftment of Human CD34+ Hematopoietic Stem and Progenitor Cells in Rhesus Monkeys

Significant advancements have been made in the use of noninvasive imaging techniques which includes dramatic improvements in resolution and sensitivity, and imaging systems designed specifically for animals. The non-invasive nature of these techniques allows longitudinal assessments over time, and the evaluation of protocols that could be applied in a human clinical setting. Imaging is also of great utility in situations when it is unclear where to focus the analyses, such as the challenges presented by systemic administration of transplanted cells, which can traffic to different anatomical sites. Our prior studies have shown that we can monitor gene expression in fetal and infant monkeys using reporter genes, microPET, and optical imaging. We have also shown that human mobilized peripheral blood stem cells (hPBSC) from adult male donors and male cord blood CD34+ hematopoietic stem cells (HSC) can readily engraft in fetal rhesus monkeys and persist over time. Other recent findings suggest that growth factor expansion of cord blood CD34+ HSC in vitro prior to transplant substantially increases the level of engraftment, with evidence of human hematopoietic cells in the rhesus host at levels significantly greater than the approximate 2% we previously reported. Our studies have also focused on new ways to use optical imaging to monitor transplanted hPBSC, cord blood HSC, and CD34+ precursors differentiated from human embryonic stem cells (hESC) that express firefly luciferase and a drug resistance gene, and the development of PET techniques for short-term tracking of transplanted cells. Transduced cells (1×104–20×106 cells/fetus) were transplanted intraperitoneally into fetal monkeys in the late first trimester under ultrasound guidance (N=27), then animals were monitored sonographically during gestation, and delivered by cesarean-section at term. At 1 week postnatal age and monthly thereafter animals were imaged for firefly luciferase expression (bioluminescence) following the intravenous injection of 100 mg/kg D-luciferin, and after the collection of blood and bone marrow for analysis. While evidence of the human Y chromosome (hSRY, hTSPY) was found in peripheral blood mononuclear cells and CD34+ immunoselected cells from marrow by qRT-PCR, the range of copies detected over time did not reflect the imaging findings. Foci of engrafted human cells were found to persist for ≥1 year, to date, in the rhesus host and in anatomical areas not previously identified (e.g., ribs, sternum, liver). In addition, 64Cu-PTSM radiolabeled hPBSC from the same donors were injected postnatally and cell trafficking monitored without evidence of toxicity or adverse effects. These in vivo imaging studies have provided unique insights into the fate of CD34+ cells post-transplantation, and indicate the importance of these techniques for cell transplant protocols.

Identification of a rare subset of adipose tissue-resident progenitor cells, which express CD133 and TRPC3 as a VEGF-regulated Ca 2+ entry channel

VEGF-induced Ca 2+ signalling was investigated in CD133 +/VEGFR-2 + progenitor cells isolated from human adipose stroma. Colonies derived from CD133 + immunoselected cells displayed inhomogenous Ca 2+ signals, with variable magnitude of VEGF-induced Ca 2+ entry, which positively correlated with expression of the Ca 2+ channel protein TRPC3. High levels of VEGF-induced Ca 2+ entry and TRPC3 expression were preferentially detected in rim areas of expanding colonies. Dominant negative suppression of TRPC3 inhibited VEGF-induced Ca 2+entry into CD133 + cells. Our results identify TRPC3 as a key Ca 2+ entry channel in a subset of CD133 + stem cells. We suggest TRPC3 as an essential determinant of cell fate in CD133 + progenitor-derived colonies.

CMRF-56 Immunoselected Blood Dendritic Cell Preparations Activated With GM-CSF Induce Potent Antimyeloma Cytotoxic T-cell Responses

The efficient antigen-presenting function of dendritic cells (DC) makes them an attractive cellular adjuvant for clinical immunotherapeutic protocols aimed at eradicating minimal residual disease after conventional treatment of multiple myeloma (MM) and other malignancies. We used single-step positive immunoselection with biotinylated CMRF-56 monoclonal antibody to generate a CD11c blood DC (BDC) enriched antigen-presenting cell population, which, after exposure to activation stimuli for as little as 2 hours, displayed a mature costimulatory BDC phenotype and secreted inflammatory cytokines. Of the activation stimuli tested, granulocyte macrophage colony-stimulating factor (GM-CSF) provided optimal activation of the CMRF-56 immunoselected preparations and primed efficient cytotoxic T cell (CTL) responses using MART-1 peptide as a model tumor-associated antigen. In addition, GM-CSF activated CMRF-56 immunoselected cells cross-presented MM cell lysate and improved the MM-specific polyclonal CTL response (no activation 18.8%+/-4.3% vs. GM-CSF activation 40.9%+/-7.3%, P=0.051). CMRF-56 immunoselected BDC migrated in vitro both spontaneously and specifically toward the secondary lymphoid chemokine CCL21. Their migration was also significantly improved by GM-CSF and prostaglandin E2 activation and a greater percentage of activated BDC migrated specifically compared with monocyte-derived DC. These results indicate that the CMRF-56 immunoselected BDC preparations can cross-present antigen for effective anti-MM CTL responses and that limited exposure to maturation stimuli can produce phenotypically and functionally mature migrating DC. CMRF-56 immunoselected cells are suitable for use as part of an immunotherapeutic anti-MM vaccine.

Isolation of immunoresistant human glioma cell clones after selection with alloreactive cytotoxic T lymphocytes: cytogenetic and molecular cytogenetic characterization

Intratumoral heterogeneity and genetic instability within gliomas may allow intrinsically immunoresistant (IR) cells to escape alloreactive cytotoxic T lymphocyte (aCTL) cellular immunotherapy. The potential existence of aCTL-resistant variants prompted us to investigate whether cellular immunotherapy resistant glioma models could be isolated. To generate the models, repeated intermittent or continuous selective pressure (ISP or CSP) with multiple aCTL populations was applied to a low-passage glioblastoma cell explant, 13-06-MG, obtained from a patient at initial diagnosis. IL-6 and IL-8 secretion was greater in coincubates of aCTL cells with 13-06-ISP and 13-06-CSP immunoselected cells than those with 13-06-MG parental cells. Initially, the immunoselected cells were less sensitive to aCTL lysis; however, the reduced aCTL-sensitivity was not maintained upon further selection. We therefore isolated IR clones from continuously immunoselected cells (13-06-CSP). The frequency of IR clones was 1–6 cells per 10,000 immunoselected cells. Two clones selected for further study, 13-06-IR29 and 13-06-IR30, resisted aCTL lysis in the absence of immunoselective pressure. Cytogenetic analyses revealed structural anomalies and genomic imbalances unique to the IR clones. Based on these findings, a hypothetical model is proposed that traces the origin of the IR clones to a clonal variant within the 13-06-CSP and 13-06-MG populations.

Cytotoxic T cell targeting of TRP-2 sensitizes human malignant glioma to chemotherapy

Tyrosinase-related protein (TRP)-2 is not only expressed on glioma cells, but is naturally processed and presented by their surface MHC molecules and is recognized by TRP-2-specific cytotoxic T cells. After active immunotherapy, we detected TRP-2-specific cytotoxic T lymphocyte (CTL) activity in patients' peripheral blood mononuclear cells (PBMC). Tumor cells from postvaccination resections showed significantly lower TRP-2 expression and higher sensitivity to carboplatin and temozolomide than those autologous cell lines from prevaccination resections in two patients who demonstrated CTL response to TRP-2. One of two patients underwent treatment with temozolomide after recurrence and responded dramatically. TRP-2-transfected cell line (TRP-2-U373) resulted in significant drug resistance to carboplatin and temozolomide compared to wild-type U-373 (W-U373). There was no significant difference, however, in the mRNA expression of other common drug resistance related proteins, such as BCRP-1, MGMT, MDR-1, MRP-1 and MRP-3, after TRP-2 transfection. TRP-2-U373 tumor cells were immunoselected by a TRP-2-specific CTL line. The immunoselected cells (IS-TRP-2-U373) demonstrated significantly increased sensitivity to carboplatin and temozolomide compared to TRP-2-U373. For the first time, we provide evidence that immunological targeting of tumor-associated antigen TRP-2 significantly increases sensitivity to chemotherapy.

Impact of Leukapheresis Cell Composition on Immunomagnetic Cell Selection with the Baxter Isolex 300i Device: A Statistical Analysis

Immunomagnetic cell selection (ICS) of CD34(+) cells is increasingly adopted in allogeneic and autologous transplant settings. Because many variables can affect the final results of ICS, we focused our study toward the influence exerted by the leukapheresis (LKF) cell composition on recovery, purity, and log of T and B depletion of the immunoselected cells. A total of 39 consecutive CD34(+) ICS were performed with the Isolex 300i (Baxter) device on 39 LKF from 9 HLA haploidentical donors and 20 patients. Flow cytometric analysis was performed both on the leukapheresis content and on the immunoselected cells. The statistical analysis was performed utilizing the Pearson's correlation test and the Mann-Whitney U test. The median purity and recovery of the immunoselected CD34(+) cells were 95.3% (IR: 93.0-99.0) and 55.1% (IR: 41.8-68.2), respectively. The median log of T and B depletion were 3.87 (IR: 3.5-4.3) and 2.9 (IR: 2.5-3.5), respectively. Our data indicate that not only the CD34(+) cell load but also the ratio among the cells belonging to the starting fraction can influence the results of ICS. LKF collection protocols have to be addressed to collect an high number of CD34(+) cells (>500 x 10(6)) without taking care of the contaminating cells when the Baxter Isolex 300i device is employed.

Impact of Leukapheresis Cell Composition on Immunomagnetic Cell Selection with the Baxter Isolex 300i Device: A Statistical Analysis

Immunomagnetic cell selection (ICS) of CD34+ cells is increasingly adopted in allogeneic and autologous transplant settings. Because many variables can affect the final results of ICS, we focused our study toward the influence exerted by the leukapheresis (LKF) cell composition on recovery, purity, and log of T and B depletion of the immunoselected cells. A total of 39 consecutive CD34+ ICS were performed with the Isolex 300i (Baxter) device on 39 LKF from 9 HLA haploidentical donors and 20 patients. Flow cytometric analysis was performed both on the leukapheresis content and on the immunoselected cells. The statistical analysis was performed utilizing the Pearson's correlation test and the Mann-Whitney U test. The median purity and recovery of the immunoselected CD34+ cells were 95.3% (IR: 93.0-99.0) and 55.1% (IR: 41.8-68.2), respectively. The median log of T and B depletion were 3.87 (IR: 3.5-4.3) and 2.9 (IR: 2.5-3.5), respectively. Our data indicate that not only the CD34+ cell load but also the ratio among the cells belonging to the starting fraction can influence the results of ICS. LKF collection protocols have to be addressed to collect an high number of CD34+ cells (<500 × 106) without taking care of the contaminating cells when the Baxter Isolex 300i device is employed.

Characterization of multiple CD34+ cell populations in cord blood.

Unlike granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood, which show a single homogeneous population of CD34(+) cells, umbilical cord blood (CB) CD34(+) cells are present as multiple populations, CD34(regular) and CD34(bright) (the latter comprising 7.0-58.2% of the total CD34(+) cells), using the ProCOUNT trade mark procedure or with anti-CD34 labeling of immunoselected cells. The CD34(regular) population contains cells with high forward scatter (CD34(regular)FSC(high)) and with low forward scatter (CD34(regular) FSC(low)). Immunomagnetically selected CD34(+) cells, sorted into CD34(regular), CD34(regular) FSC(high), CD34(regular)FSC(low), and CD34(bright) cell populations, were used in in vitro assays: only the CD34(regular)FSC(high) population transmigrated and showed growth of colony-forming unit (CFU) and long-term culture initiating cells (LTC-IC) colonies. The absolute number of CD34(+) cells in CB samples was determined by ProCOUNT trade mark and Stem Kit trade mark enumeration protocols. In liquid stored CB units, ProCOUNT trade mark and Stem Kit trade mark count differences are accounted for by the enumeration of CD34(bright) cells. Differences between ProCOUNT trade mark and Stem Kit trade mark counts using cryopreserved/thawed samples are accounted for by increased CD34(regular) FSC(low) cell numbers (2.0 +/- 1.4% in liquid stored and 27.8 +/- 14.6% in cryopreserved/thawed samples). The ProCOUNT trade mark assay includes the nonfunctional CD34(bright) and CD34(regular)FSC(low) cells as part of the CD34(+) cell count, thereby elevating the absolute number of CD34(+) cells. Using the Stem Kit trade mark assay method of gating, CD34(bright) and CD34(regular)FSC(low) cells are not counted. Our data indicate that the CD34(regular)FSC(high) cell population has functional characteristics based on the in vitro assays and a more accurate count of these cells can be achieved using the Stem Kit trade mark assay.

CD34+ immunoselected cells for poor graft function following allogeneic BMT

BackgroundPoor graft function without signs of graft rejection following allogeneic BMT (allo-BMT) occurs in around 9% of patients. A high incidence of hazardous complications may be encountered, leading to life-threatening situations. MethodsWe describe three patients who underwent allo-BMT for acute leukemia in first complete remission and untreated myelodysplastic syndrome. The three patients experienced prolonged and profound granulocytopenia, anemia and thrombocytopenia, despite growth factors and transfusions. This was not corrected by donor leukocytes infusion. They received a boost of CD34+ positively-selected cells from their HLA-identical sibling donors. ResultsA rapid improvement of peripheral blood cell counts was observed in both patients who were in full donor chimerism status at time of boost infusion, whereas the patient with mixed chimerism did not show any signs of improvement. Neither patient suffered further exacerbation of GvHD. DiscussionAllogeneic positively-immunoselected CD34+ cells can represent an interesting alternative treatment for poor graft function following allo- BMT, in the absence of graft rejection signs.

The alpha1 subunit of laminin-1 promotes the development of neurons by interacting with LBP110 expressed by neural crest-derived cells immunoselected from the fetal mouse gut.

A plasmalemmal protein, LBP110, which binds to the alpha1 chain of laminin-1, is acquired by the neural crest-derived precursors of enteric neurons after they colonize the gut. We tested the hypothesis that laminin-1 interacts with LBP110 to promote enteric neuronal development. The effects of laminin-1 on neuronal development were studied in cultures of cells immunoselected from fetal mouse gut (E14-15) with antibodies to LBP110 or p75NTR, a marker for enteric crest-derived cells. No matter which antibody was used, the development of cells expressing neuronal markers was increased three- to fourfold by culturing the cells on a laminin-1-containing substrate. To determine whether this effect of laminin-1 is due to the selective adherence of a neurocompetent subset of precursors, immunoselected cells were permitted to preadhere to poly-D-lysine. Addition of soluble laminin-1 24 h later promoted neuronal but not glial development. The laminin-1-induced increment in neuronal development was abolished both by a peptide containing the sequence of the LBP110-binding domain, IKVAV, and by antibodies to laminin alpha1 that recognize the IKVAV domain. Neither reagent affected the total number of cells. In contrast, the response to laminin-1 was not affected by control peptides, preimmune sera, or antibodies to laminin beta1. Laminin-1 transiently induced the expression of nuclear Fos immunoreactivity; this action was blocked specifically by the IKVAV peptide. These data are consistent with the hypothesis that LBP110 interacts with the IKVAV domain of laminin alpha1 to promote the differentiation of neurons from enteric crest-derived precursors.