Leukapheresis Samples Research Articles

Increased Levels of Myeloid-Derived Suppressor Cells (MDSCs) in Chronic Myeloid Leukemia and the Effect of Tyrosine Kinase Inhibitors on MDSCs in Vitro

Abstract 2744 BackgroundMyeloid-derived suppressor cells (MDSCs) are cells of myeloid origin that are able to inhibit immune cells such as T cells. MDSCs have been found in many different solid tumors and the role of these cells in cancer is currently in focus since they hamper anti-tumor immune responses and thereby lead to a worse prognosis for the patients. The presence and function of MDSCs in hematological malignancies such as chronic myeloid leukemia (CML) has so far not been extensively studied. Since patients with CML have malignant immature myeloid cells in blood and bone marrow it is of interest to investigate if these cells are myeloid suppressor cells. Furthermore, the level of MDSCs in solid tumors decreases after treatment with the tyrosine kinase inhibitor (TKI) sunitinib. CML patients are treated with other TKIs such as imatinib and dasatinib and it is of interest to investigate if these have the same effect on MDSCs. Imatinib and dasatinib are intended to block bcr-abl in the tumor cells. In addition to this, these TKIs have been shown to interact with other tyrosine kinases present in immune cells. AimThe aim of the study was to investigate the presence of MDSCs and the MDSC-associated molecule Arginiase-1 in CML patients and cell lines as well as to investigate the effect of the TKIs on MDSCs in vitro. MethodsCryopreserved leukapheresis samples from newly diagnosed patients in chronic phase (n=18) from high (n=11) and low (n=7) Sokal score risk group CML patients and buffy coats from healthy controls (HC, n=21) as well as CML cell lines BV173, K562 and CML-T1 were investigated for the presence of MDSCs (CD11b+CD14-CD33+ cells) by flow cytometry. Peripheral blood mononuclear cells (PBMCs) from healthy donors were cultured in different concentrations of imatinib or dasatinib and the level of MDSCs in cultures was assessed after 2 and 4 days of culture by flow cytometry. The level of the MDSC-associated molecule Arginase-1 in cells from CML patients (n=5), controls (n=5) and CML cell lines as well as in healthy PBMCs after culture with imatinib or dasatinib was assessed by real time PCR. ResultsCML patients had significantly higher levels of MDSCs compared to health controls (medians 2,6% in CML and 0,8% in HC). When CML patients were divided into Sokal high and low risk groups the high risk group had higher MDSC level (median 3,6%) compared to the low risk group (median 0,8%). The level of MDSC in Sokal low risk group did not differ from the level in HCs. Arginase-1 is a molecule linked to the immune inhibitory effects of MDSCs. The mRNA expression level of Arginase-1 was higher in CML patients compared to HCs as assessed by real time PCR. When healthy PBMCs were cultured with imatinib or dasatinib for 2 to 4 days, the level of MDSCs increased in a dose dependent manner. In accordance with this, the Arginase-1 mRNA expression also increased with treatment in a dose dependent manner. All cell lines investigated had cells with the MDSC phenotype, however not all cells exhibited that phenotype. Only K562 expressed Arginase-1 and the expression increased when the cells were treated with imatinib or dasatinib. ConclusionsCML have higher MDSC levels compared to healthy individuals and the level correlates to Sokal score. The level of MDSCs also correlates with mRNA expression of Arginase-1. Interestingly, treatment of healthy donor PBMCs with the TKIs imatinib or dasatinib increases the level of MDSCs in contrast to results shown with sunitinib. Disclosures:Simonsson:Novartis, BMS, Merck, Pfizer: Consultancy, Honoraria.

Mesenchymal stem cells derived from bone marrow and leukapheresis show different putative subpopulations

Mesenchymal stem cells (MSCs), with their high proliferative and differentiation potential, in addition to their plasticity, are promising candidates for cell therapeutic applications. Bone marrow (BM) harvest is still the main source of MSCs in spite of being traumatic and painful. Clinical indications for peripheral blood-derived MSCs are rapidly increasing. This study was done to compare the biological properties of MSCs derived from BM and leukapheresis product regarding viability, fold expansion, cell cycle status and putative subpopulations. MSCs were isolated and cultured from BM and leukapheresis samples after stem cell mobilization. MSCs were characterized by morphology and immunophenotyping. Their viability, fold expansion and cell cycle status were compared. Estimation of putative cells among the mesenchymal population was done by dual expression of CD44 and Oct4. Leukapheresis derived MSCs were found to be comparable to BM-MSCs regarding their viability, fold expansion and cell cycle status, however they differ in their putative subpopulations. BM samples had significantly higher percentage of putative population than leukapheresis samples (18.38± 3.21% vs 5.43± 1.26%, P=0.009). These results indicated the possible isolation and expansion of MSCs from leukapheresis samples. The lower putative subpopulations among leukapheresis derived MSCs may be due to lack of BM microenvironment related factors needed to maintain pluripotency or due to the current methods of cell mobilization that have been optimized for hematopoietic stem cells rather than MSCs.

BCL6 Is Required for the Maintenance of Leukemia-Initiating Cells In Chronic Myeloid Leukemia

AbstractAbstract 202 Background:Chronic myeloid leukemia (CML) can be effectively treated for many years with tyrosine kinase inhibitors (TKI). However, unless CML patients take TKI-treatment life-long, leukemia will eventually recur, which is attributed to the failure of TKI-treatment to eradicate leukemia stem cells in CML. Relapse from leukemia stem cells in CML often results in TKI-resistant blast crisis, which is fatal within months. Approach:Recent work demonstrated that FoxO3A is critical for maintenance of leukemia stem cells in chronic myeloid leukemia (CML). The mechanism of FoxO3A-dependent maintenance of leukemia stem cells remained unclear. Here we identified the BCL6 protooncogene downstream of FoxO3A as a critical effector molecule of self-renewal signaling in CML-initiating cells. BCL6 is known as a proto-oncogene in Diffuse Large B cell Lymphoma (DLBCL), where it functions as transcriptional repressor of p53. BCL6-Null CML cells fail to initiate leukemia:Studying gene expression changes of CML cells in 6 patients before and after treatment with Imatinib, we found that BCL6 mRNA levels were increased by >15-fold in response to Imatinib-treatment. Studying CD34+ CD38- CML cells from leukapheresis samples of two patients and CML cell lines, we found that overnight incubation with Imatinib resulted in a >12-fold increase of BCL6 expression at the mRNA and protein level. Previous studies showed that FoxO factors are required for transcriptional activation of BCL6 and FoxO3A was recently identified as a critical factor of leukemia stem cell maintenance in CML. Here we showed that inducible activation of FoxO3A indeed leads to de novo expression of BCL6 in human CML cells. We next tested the functional significance of BCL6 expression in CML cells in a genetic experiment. To this end, we used a classical mouse model for CML-like leukemia and transformed Lin− Sca-1+ c-kit+ (LSK) cells from BCL6+/+ and BCL6-Null mice with BCR-ABL1. While CML transformation efficiency was similar for BCL6+/+ and BCL6-Null LSK cells, the LSK phenotype was rapidly lost in BCL6-Null CML cells. To elucidate the mechanism of progressive loss of LSK cells in BCL6-Null CML, we performed a systematic analysis of gene expression changes in BCL6+/+ and BCL6-Null CML cells: The ABCG2 transporter, which is required for the side population (SP+) phenotype in LSK cells was reduced by >7-fold in the absence of BCL6. SP+ LSK cells represent a highly drug resistant CML subpopulation with leukemia-initiation capacity. In addition, BCL6-Null CML cells express excessively high levels of p53 at the protein level. A genome-wide mapping approach of BCL6-DNA interactions using ChIP-seq showed that BCL6 strongly binds to and represses the p53 promoter. Importantly, BCL6 expression represents a critical requirement for CML cells to form colonies in semisolid agar. Compared to BCL6+/+ CML cells (>400 colonies/10,000 cells), colony formation by BCL6-Null CML cells was reduced by ≂f100-fold (<5 colonies). After an initially successful engraftment of BCL6-Null CML cells in NOD/SCID mice (as visualized by luciferase bioimaging), BCL6-Null CML cells failed to initiate leukemia, whereas NOD/SCID mice injected with BCL6+/+ CML cells succumbed to the disease. Targeted inhibition of BCL6 for leukemia stem cell eradication in CML:A retro-inverso BCL6 peptide inhibitor (RI-BPI) was recently developed for targeted therapy of DLBCL. Therefore, we tested the potential therapeutic usefulness of RI-BPI for eradication of BCL6-dependent leukemia-initiating cells. Human CML cells were incubated overnight in the presence of 5 μ mol/l RI-BPI or vehicle control. While Hoechst 33342 staining revealed a distinct side population of ≂f2.8% CML cells, overnight incubation with RI-BPI reduced the frequency of ABCG2+ side population cells by more than 30-fold. Likewise, RI-BPI incubation decreased colony formation by >5-fold in human CML cells. Importantly, RI-BPI treatment of CML cells resulted in significantly prolonged overall survival of xenografted NOD/SCID mice and decreased penetrance of leukemia (10 mice/group; p=0.032), Conclusion:Pharmacological inhibition of BCL6 represents a powerful strategy to eradicate leukemia-initiating cells in CML. Clinical validation of this concept could limit the duration of TKI-treatment in CML patients, which is currently life-long, and substantially decrease the risk of blast crisis transformation. Disclosures:Hochhaus:Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Efficient clinical-scale enrichment of lymphocytes for use in adoptive immunotherapy using a modified counterflow centrifugal elutriation program

Clinical-scale lymphocyte enrichment from a leukapheresis product has been performed most routinely using costly magnetic bead separation systems that deplete monocytes, but this procedure may leave behind residual beads or antibodies in the enriched cell product. Counterflow centrifugal elutriation has been demonstrated previously to enrich monocytes efficiently for generation of dendritic cells. This study describes a modified elutriation procedure for efficient bead-free economical enrichment of lymphocytes from leukapheresis products from healthy donors and study subjects with human immunodeficiency virus (HIV) infection or malignancy. Modified program settings and conditions for the CaridianBCT Elutra device were investigated to optimize lymphocyte enrichment and recovery. Lymphocyte enrichment was measured using a novel approach utilizing cell sizing analysis on a Beckman Coulter Multisizer and confirmed by flow cytometry phenotypic analysis. Efficient enrichment and recovery of lymphocytes from leukapheresis cell products was achieved using modified elutriation settings for flow rate and fraction volume. Elutriation allowed for enrichment of larger numbers of lymphocytes compared with depletion of monocytes by bead adherence, with a trend toward increased lymphocyte purity and yield via elutriation, resulting in a substantial reduction in the cost of enrichment per cell. Importantly, significant lymphocyte enrichment could be accomplished using leukapheresis samples from healthy donors (n=12) or from study subjects with HIV infection (n=15) or malignancy (n=12). Clinical-scale closed-system elutriation can be performed efficiently for the selective enrichment of lymphocytes for immunotherapy protocols. This represents an improvement in cost, yield and purity over current methods that require the addition of monocyte-depleting beads.

Innate immune modulation as a mechanism for therapeutic action of early EPCs in a nude rat model of pulmonary hypertension

Early and late‐outgrowth endothelial progenitor cells (EPCs) were cultured from human leukapheresis samples and tested for therapeutic efficacy in a nude rat, monocrotaline (MCT)‐induced model of pulmonary hypertension (PH). Three days after injection of MCT, 106 cells were administered to nude rats via central venous injection. Three weeks after cell injection, right ventricular systolic pressure was elevated in MCT treated animals (61 ± 3.8 vs. 27 ± 0.5 mmHg no MCT control). The administration of early‐EPCs prevented the onset of PH (33 ± 3.2 mmHg), while delivery of late‐EPCs produced no improvement (60 ± 4.0 mmHg). Analysis of cell retention by histology and PCR revealed that, despite the therapeutic benefit of early‐EPCs, neither cell type was retained within the lung at 24 hours post injection. A subsequent in vitro characterization of early‐EPCs revealed a capacity to modulate the innate immune response in a manner similar to that of dendritic cells. Early‐EPCs, which are positive for CD14, MHC II, CD86 and CD11c, and negative for CD1a and CD83, increased surface expression of CD86 and CD83 and decreased surface CD14 in response to stimulation with LPS. Exposure to LPS also induced the secretion of IL‐10 but not IL‐12. As with dendritic cells, co‐culture of early‐EPCs with autologous natural killer (NK) cells in the presence of LPS induced NK cytotoxicity, suggesting a potential mechanism of therapeutic action.

Inability to Mediate Prolonged Reduction of Regulatory T Cells After Transfer of Autologous CD25-depleted PBMC and Interleukin-2 After Lymphodepleting Chemotherapy

CD25CD4 regulatory T cells (Treg) regulate peripheral self-tolerance and possess the ability to suppress antitumor responses, which may explain the poor clinical response of cancer patients undergoing active immunization protocols, and provides the rationale for neutralizing Treg cells in vivo to strengthen local antitumor immune responses. Because interleukin-2 (IL-2) mediates tumor regression in about 15% of treated patients but simultaneously increases Treg cells, we hypothesized that transient elimination of Treg cells will enhance the clinical effectiveness of IL-2 therapy. In the current study, 5 patients with metastatic melanoma who were refractory to prior IL-2 received a lymphodepleting preparative regimen followed by the adoptive transfer of autologous lymphocytes depleted of CD25 Treg cells and high-dose IL-2 administration. CD25 cells were eliminated from patient leukapheresis samples using a clinical-grade, large-scale immunomagnetic system, leaving CD8 and CD25CD4 T cells intact. In the early aftermath of CD25 Treg cell-depleted cell infusion, CD25FOXP3+ CD4 Treg cells rapidly repopulated the peripheral blood of treated patients with 18% to 63% of CD4 T cells expressing FOXP3. Recovering CD25CD4 T cells exhibited suppressive activity against CD25CD4 effector T-cell proliferation in vitro. No patient experienced objective tumor regression or autoimmunity. Our results indicate that in vivo transfer of autologous CD25-depleted mononuclear populations to lymphopenic patients in combination with high-dose IL-2 is not sufficient to mediate prolonged reduction of Treg cells after IL-2 administration.

Blood progenitor cell separation from clinical leukapheresis product by magnetic nanoparticle binding and magnetophoresis

Positive selection of CD34+ blood progenitor cells from circulation has been reported to improve patient recovery in applications of autologous transplantation. Current magnetic separation methods rely on cell capture and release on solid supports rather than sorting from flowing suspensions, which limits the range of therapeutic applications and the process scale up. We tested CD34+ cell immunomagnetic labeling and isolation from fresh leukocyte fraction of peripheral blood (leukapheresis) using the continuous quadrupole magnetic flow sorter (QMS), consisting of a flow channel (SHOT, Greenville, IN) and a quadrupole magnet with a maximum field intensity (B(o)) of 1.42 T and a mean force field strength (S(m)) of 1.45 x 10(8) TA/m(2). Both the sample magnetophoretic mobility (m) and the inlet and outlet flow patterns highly affect the QMS performance. Seven commercial progenitor cell labeling reagent combinations were quantitatively evaluated by measuring magnetophoretic mobility of a high CD34 expression cell line, KG-1a, using the cell tracking velocimeter (CTV). The CD34 Progenitor Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) showed the strongest labeling of KG-1a cells and was selected for progenitor cell enrichment from 11 fresh and 11 cryopreserved clinical leukapheresis samples derived from different donors. The CD34+ cells were isolated with a purity of 60-96%, a recovery of 18-60%, an enrichment rate of 12-169, and a throughput of (1.7-9.3) x 10(4) cells/s. The results also showed a highly regular dependence of the QMS performance on the flow conditions that agreed with the theoretical predictions based on the CD34+ cell magnetophoretic mobility.

Quality determination of magnetic labeling reagents by cell magnetophoresis measurement

Access to high affinity, high magnetic susceptibility antibody-nanoparticle combinations specific to target cell populations is important for isolation of low frequency cells or cells expressing low level of antigenic markers. A specialized instrument (Cell Tracking Velocimeter, CTV) to measure single cell magnetophoretic mobility (MM) on large cell sample sizes (in thousands) was developed in our laboratories to determine effects of magnetic cell labeling. First, using a hematopoietic progenitor cell line, KG-1a (ATCC, Manassas, VA) seven different CD34 monoclonal antibody and nanoparticles combinations were tested for highest MM: Progenitor KitTM (Miltenyi Biotec, CA) showed the highest MM (2.14±0.31 mm3/T-A-s with 95% confidence). Second, the specificity of the Kit for labeling of progenitor cells in clinical leukapheresis samples was determined by comparing CTV-measured frequency of cells moving in the magnetic field with the flow cytometry (FCM) measured frequency of cells expressing CD34 marker (n=6, r2=0.87). Third, clinical leukapheresis samples labeled with the Kit were enriched for CD34+ cells by continuous magnetophoresis and assayed by CTV and FCM. The results show that the cell MM and the performance of the magnetic separation protocol depend on selection of the magnetic labeling reagent.

Growth Factor Receptors in Hematopoietic Stem Cells: EPH Family Expression in CD34+ and CD133+ Cell Populations from Mobilized Peripheral Blood

Cell-surface antigen expression of hematopoietic stem cells has a crucial role in characterizing cell subpopulation with distinct functional properties. The Eph receptors are the largest receptor tyrosine kinase family being involved in processes like vascular remodelling during development and physiological and pathological angiogenesis. Some Eph/Ephrin members are expressed in hematopoietic cells. The ability to isolate purified cell populations co-expressing CD34 and CD133 antigens as most commonly used markers for identification of hematopoietic progenitors has provided the opportunity to identify their surface-receptor profile. As positively expressed CD34 and CD133 cells take place not only in hematopoietic but also in endothelial differentiation, we aimed to define the Eph/Ephrin characteristic of these cells and relate these findings to new therapy strategies. Positive selections of CD34 and CD133 cells from PBPC in lymphoma patients were performed using magnetic beads and AutoMACS (Miltenyi Biotec) device. The purity of isolated cells was tested by flow cytometry. Immunocytochemistry was used to assess the Eph/Ephrin expression profile of positively selected samples. Our study revealed that all samples (10 from CD34+ and 8 from CD133+ cells) expressed one or more of Eph/Ephrin antigens in different proportions. All CD34+ cell samples, and 6 of 8 in the CD133+ cell fraction were strongly immunoreactive for EphA2. EphB2 was strongly expressed in all CD133+ cases, but 50% of the CD34 positive group lacked or weakly expressed this receptor. EphB4 was negative in 9 of 10 CD34+ cases and in all CD133+ cells. Thus, we have shown the surface marker profile of positively selected CD34 and CD133 cells in leukapheresis samples from lymphoma patients with regard to Eph/Ephrin receptors and discussed their biological clinical potential.

Large-Scale Depletion of CD25+ Regulatory T Cells from Patient Leukapheresis Samples

The ability to selectively enrich or deplete T lymphocytes of specific phenotype and function holds significant promise for application in adoptive immunotherapy protocols. Although CD4+ T cells can have an impact on CD8+ T-cell effector function, memory, and maintenance, a subset of CD4+ T cells, CD25+ regulatory T cells (Treg), can regulate peripheral self tolerance and possess the ability to suppress antitumor responses. The authors report the ability to selectively deplete CD25+ Treg cells from patient leukapheresis samples using a clinical-grade, large-scale immunomagnetic system. Using leukapheresis samples containing up to 1.3 x 10(10) white blood cells, efficient depletion of Treg cells was measured by flow cytometric analysis of CD25 expression and FOXP3 expression on post-depletion products. Remnant CD25+ cells could not be detected in CD25-depleted products after short-term culture in IL-2 or enriched following secondary immunomagnetic selection for CD25+ cells, confirming that efficient depletion had occurred. In parallel to efficient enrichment of CD25- cells, immunomagnetic selection resulted in the recovery of Treg cells, since CD25+ lymphocytes removed during depletion were primarily composed of CD4+ T cells that expressed high levels of FOXP3 and possessed suppressive activity against autologous TCR-stimulated CD4+ CD25- T cells in vitro. These results show that selective separation of functional CD25+ Treg cells from large-scale samples can be performed in large scale under clinical-grade conditions with sufficient selection, recovery, viability, ability to expand, and function for potential use in adoptive immunotherapy.

Génération in vitro de globules rouges humains matures et fonctionnels : un modèle d’étude aux perspectives multidisciplinaires

We describe a technical approach permitting massive expansion of CD34+ stem cells (up to 1.95 x 10(6)-fold) and their full ex vivo conversion into mature red blood cells (RBCs). This three-step protocol can be adapted to hematopoietic stem cells (HSC) of various origins. First, cell proliferation and erythroid differentiation are induced in serum-free media supplemented with stem cell factor, interleukin-3 and erythropoietin (Epo) for 8 days. The cells are then co-cultured with either the murine stromal cell line MS-5 or human mesenchymal cells for 3 days in the presence of Epo alone. Finally, all exogenous factors are withdrawn and the cells are incubated on a simple stroma for up to 10 days. The ex vivo microenvironment strongly influences both the terminal maturation of erythroid cells and hemoglobin (Hb) synthesis. Critically, in vitro-generated RBCs have all the characteristics of functional native adult RBCs in terms of their enzyme content, membrane deformability, and capacity to fix and release oxygen. In addition, their behavior in the murine NOD/SCID model mirrors that of native RBCs. This new concept of "cultured RBCs" (cRBC) has major implications for basic research on terminal erythropoiesis and for patient management. Currently, the potential yield of functional red cells is compatible with clinical requirements, as several units of packed RBCs can be produced from a single donation. Importantly, infused cRBC would all have a life-span of about 120 days, whereas the mean half-life of normal donor RBCs is only 28 days. This would help to minimize the transfusion exposure of patients requiring regular treatment, thereby reducing the risk of iron overload and allo-immunization. The use of autologous CD34+ cells isolated from leukapheresis samples could be beneficial for patients who no longer tolerate allogeneic RBCs. This new method should also prove useful for analyzing the mechanisms of terminal erythropoiesis, including hemoglobin synthesis. Finally, it could provide a tool for investigating the lifecycle of blood parasites such as Plasmodium, the agent of malaria.

Selective cytotoxicity and telomere damage in leukemia cells using the telomerase inhibitor BIBR1532

AbstractTelomerase represents an attractive target for a mechanism-based therapeutic approach because its activation has been associated with unlimited proliferation in most cancer cells. Recently, a nonnucleosidic small molecule inhibitor, BIBR1532 (2-[(E)-3-naphtalen-2-yl-but-2-enoylamino]-benzoic acid), has been identified that is highly selective for inhibition of telomerase, resulting in delayed growth arrest of tumor cells. Here we examined the effects of BIBR1532 in different leukemia cell lines as well as in primary cells from patients with acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL) in short-term culture assays. We observed a dose-dependent direct cytotoxicity in concentrations ranging from 30 to 80 μM. Interestingly, cell death was not dependent on the catalytic activity of telomerase but was delayed in cells with very long telomeres. We observed time-dependent individual telomere erosion, which was associated with loss of telomeric repeat binding factor 2 (TRF2) and increased phosphorylation of p53. Importantly, the proliferative capacity of normal CD34+ cells from cord blood and leukapheresis samples was not affected by treatment with BIBR1532. We conclude that using this class of telomerase inhibitor at higher concentrations exerts a direct cytotoxic effect on malignant cells of the hematopoietic system, which appears to derive from direct damage of the structure of individual telomeres and must be dissected from telomerase-suppressed overall telomere shortening. (Blood. 2005; 105:1742-1749)

Aldehyde Dehydrogenase Activity - the Better Marker for the Quality of Hematopoietic Stem Cell Transplants?!.

The use of functional rather than cell surface markers to identify and isolate stem cells has several advantages. Recently, aldehyde dehydrogenase (ALDH) enzyme activity has been shown to be a metabolic marker for primitive hematopoietic progenitor cells. We compared 7-day-expression kinetics of ALDH vs. the standard marker in human hematopoietic stem cell transplantation, CD34, and related these data to the colony-forming ability of bone marrow and leukapheresis samples. Cell probes directly taken from whole transplants were first analyzed on the day of harvest (day 0) using flow cytometry (CD34, ALDEFLUOR®) and methyl cellulose (MC) assays. To resemble usual conditions during transport of clinical transplants, cell samples were then stored without additional modification at either +4°C or room temperature (RT). Relative CD34+, ALDH+ and CFU numbers were determined at various time points (days 1, 2, 3 and 7). During this time course we found that progenitor (CFU-GM) cell activity correlated very well with ALDH enzyme function but not with CD34 expression, the commonly used parameter to evaluate the quality of hematopoietic stem cell transplants. This indicates that ALDH expression may be a more reliable marker for quality control of HSC transplants. Based on our assay systems we also could show that PBSC transplants lose most of their CFU-activity (>80%) within just 48 hours if stored at room temperature, but are stable at +4°C. Thus, transport of PBSC transplants at RT should be strongly avoided. In conclusion, our data may have important implications for both quality control and transport organization of HSC transplants, especially given the permanent increase in numbers of PBSCT from matched unrelated donors.

Mobilized peripheral blood SSCloALDHbr cells have the phenotypic and functional properties of primitive haematopoietic cells and their number correlates with engraftment following autologous transplantation.

We have developed an approach for identifying primitive mobilized peripheral blood cells (PBSC) that express high levels of aldehyde dehydrogenase (ALDH). PBSC were stained with a fluorescent ALDH substrate, termed BODIPY trade mark -aminoacetaldehyde (BAAA), and then analysed using flow cytometry. A population of cells with a low side scatter (SSC) and a high level of BAAA staining, termed the SSCloALDHbr population, was readily discriminated and comprised a mean of 3 +/- 5% of leukapheresis samples. A mean of 73 +/- 11% of the SSCloALDHbr population expressed CD34 and 56 +/- 25% of all the mobilized CD34+ cells resided within the SSCloALDHbr population. The SSCloALDHbr population was largely depleted of cells with mature phenotypes and enriched for cells with immature phenotypes. Sorted SSCloALDHbr and SSCloALDHbr CD34+ PBSC were enriched for progenitors with the ability to (1) generate colony-forming units (CFU) and long-term culture (LTC)-derived CFU, (2) expand in primary and secondary LTC, and (3) generate multiple cell lineages. In 21 cancer patients who had undergone autologous PBSC transplantation, the number of infused SSCloALDHbr cells/kg highly correlated with the time to neutrophil and platelet engraftment (P < 0.015 and P < 0.003 respectively). In summary, peripheral blood SSCloALDHbr cells have the phenotypic and functional properties of primitive haematopoietic cells and their number correlates with engraftment following autologous transplantation.

Prognostic factors influencing feasibility of cytogenetic and molecular analysis in leukapheresis products in chronic myelogenous leukemia patients.

Conventional cytogenetic (CC) study and molecular analysis were performed in 150 leukapheresis products from 36 patients diagnosed with chronic myelogenous leukemia who were included in an autologous stem cell transplantation program. The aims of the study were to evaluate the effectiveness of these two methods for the detection of residual disease in the harvest and to identify the factors influencing the number of cycling cells present in the apheresis products. Progenitor cell mobilization procedures performed late after diagnosis (>12 months), a short interval between interferon-alpha discontinuation and mobilization (<3.5 months), and an intensive mobilization regimen (idarubicin, cytarabine, and etoposide, ICE protocol) were associated with a low probability of obtaining 25 metaphases, which was achieved in only 41 instances (25% of the samples). In 38 samples, less than ten metaphases were obtained; a peripheral blood leukocyte count <1.0x10(9)/l at mobilization and mononuclear cell counts in the bag <0.5x10(8)/kg significantly increased the probability to obtain less than ten metaphases for CC analysis. Previous interferon-alpha treatment during > or =12 months and low mononuclear cell counts in the bag (<0.5x10(8)/kg) increased the probability of not obtaining mitosis for cytogenetic analysis. Molecular analysis by the polymerase chain reaction (PCR) technique did not give discriminate information in the samples not evaluable by cytogenetics due to the high frequency of PCR-positive results. We conclude that new techniques such as hypermetaphase fluorescence in situ hybridization (FISH), interphase FISH, or quantitative PCR need to be routinely employed in the study of leukapheresis samples of chronic myelogenous leukemia patients for a better assessment of the neoplastic contamination of the infused products.

Influence of the different CD34+ and CD34- cell subsets infused on clinical outcome after non-myeloablative allogeneic peripheral blood transplantation from human leucocyte antigen-identical sibling donors.

Currently, no information is available regarding the influence of the different CD34+ cell subsets infused on the haematopoietic recovery, following non-myeloablative allogeneic peripheral blood stem cell transplantation (allo-PBSCT). We have explored, in a group of 13 patients receiving non-myeloablative allo-PBSCT from human leucocyte antigen-identical sibling donors, the influence of the total dose of CD34+ haematopoietic progenitor cells (HPC) infused, compared with that of the different CD34+ HPC and CD34- leucocyte subsets in the leukapheresis samples, on both engraftment and clinical outcome. The overall numbers of total CD34+ HPC (P = 0.002) and myelomonocytic-committed CD34+ HPC infused (P = 0.0002) were strongly associated with neutrophil recovery (> 1 x 109 neutrophils/l), the latter being the only independent parameter influencing neutrophil recovery. Regarding long-term engraftment, only the number of immature CD34+ HPC infused/kg correlated with the duration of hospitalization in the first 2 years after discharge (r = -0.75, P = 0.005). Both the overall amount of CD34+ HPC and the number of myelomonocytic CD34+ HPC infused showed a significant influence on the risk of graft-versus-host disease (GVHD). Thus, the overall probability of GVHD was 100%vs 25% for patients receiving >/= 5 x 106 CD34+ HPC or >/= 3.5 x 106 of myelomonocytic-committed CD34+ HPC vs lower doses (P = 0.013). None of the other CD34+ and CD34- cell subsets analysed correlated with development of GVHD. In summary, our results suggest that in non-myeloablative allo-PBSCT, high numbers of CD34+ HPC, especially the myelomonocytic-committed CD34+ progenitors, lead to rapid neutrophil engraftment. However, they also strongly impair clinical outcome by increasing the incidence of GVHD.

Mitoxantrone/Ifosfamide/Etoposide Salvage Regimen with Rituximab for In Vivo Purging in Patients with Relapsed Lymphoma

Treatment with the anti-CD20 antibody rituximab prior to stem cell collection may lead to tumor-free stem cell collections in patients with B-cell lymphoma undergoing autologous stem cell transplantation. To test the feasibility of obtaining polymerase chain reaction (PCR)- negative stem cell collection, 30 patients with a variety of B-cell lymphomas were enrolled in a protocol employing a common MINE (mitoxantrone/ifosfamide/etoposide) salvage regimen with rituximab (in vivo purging). Rituximab 400 mg/m2 was administered weekly for 3 weeks on days 1, 6, and 8 in relation to the last MINE cycle, which was followed by growth factor–stimulated peripheral stem cell collection. The median number of CD34+ cells/kg was 2.5 million cells/kg collected over a median of 5 days. Polymerase chain reaction amplification for the t(14;18) or the heavy-chain gene rearrangement was performed prior to treatment and on the leukapheresis sample. Out of 15 patients who had a positive PCR signal prior to treatment, 10 had PCR-negative stem cell collections, whereas 5 had PCR-positive stem cell collections. After high-dose chemotherapy and stem cell transplant, all patients with a PCR-positive signal pretreatment became PCR negative. We conclude that rituximab may increase the yield of tumor-free stem cells. Higher rates of PCR negativity have been reported when more intense and protracted chemoimmunotherapy regimens have been employed. The magnitude of clinical benefit and the significance of the PCR analysis in stem cells after rituximab requires larger studies.

Large-scale monocyte enrichment coupled with a closed culture system for the generation of human dendritic cells

Conventional methods for generating monocyte-derived dendritic cells (DC) for clinical trials utilize the property of plastic adherence to select monocytes from leukapheresis samples. This method is labor-intensive and has the potential for contamination at various steps. We evaluated a large-scale monocyte enrichment procedure using a cell selector (Isolex 300i®) followed by culture in a sterile bag system (Stericell®) for generation of DC. DC generated in tissue culture flasks after monocyte selection by plastic adherence were compared to those generated in Stericell® bags after monocyte enrichment by negative selection with the Isolex® 300i. DC were matured with lipopolysaccharide and pulsed with a peptide derived from the melanoma antigen gp100. Peptide-pulsed DC cultured by the two techniques were evaluated for phenotype, viability, ability to induce allogeneic and peptide-specific autologous proliferative responses as well as peptide-specific cytotoxic T-cell responses. The mean monocyte yield from leukapheresis collections was 17±2.4%, which increased to 52±11% after Isolex® selection. The DC yield of plated mononuclear cells from flasks or bags was 2.7±0.96% and 4.84±2.65%, respectively. DC cultured by both methods expressed high levels of CD86, CD80, CD40, CD83, CD44, CD11c and CD58, and was comparable in their ability to induce allogeneic and peptide-specific autologous proliferative responses as well as gp100 peptide-specific cytotoxic T-cell responses. These results indicate that potent monocyte-derived DC can be generated in a closed culture bag system after monocyte enrichment by immunomagnetic negative selection. Due to the closed nature of the enrichment and culture systems, the potential for contamination is minimized. This protocol is well suited for culturing large numbers of DC for clinical immunotherapy trials.

Mobilization of dendritic cells from patients with breast cancer into peripheral blood stem cell leukapheresis samples using Flt-3-Ligand and G-CSF or GM-CSF.

Treatment with myeloablative chemotherapy and autologous peripheral blood stem cell (PBSC) transplantation followed by vaccination with autologous dendritic cells (DCs) treated with tumor antigens is a promising therapeutic strategy for several types of cancer. Obtaining sufficient numbers of both PBSCs and DCs is central to this approach. Previously, it has been shown that administration of Flt-3-Ligand (FL) combined with either G-CSF or GM-CSF mobilizes large numbers of PBSCs in patients with cancer. In the current study, we sought to determine whether these same cytokines could simultaneously mobilize DCs into the PBSC leukapheresis collection. DCs were analysed in PBSC leukapheresis samples obtained from five patients with high-risk breast cancer who received G-CSF alone as priming prior to leukapheresis, four patients who received FL+G-CSF and five patients who received FL+GM-CSF. DCs were defined as cells with a lin(dim/-) HLA-DR+ CD11c+ phenotype. The proportions of DCs in the FL+G-CSF and FL+GM-CSF samples were significantly higher than in pre-mobilization peripheral blood and G-CSF leukapheresis samples. The mean yield of DCs/kg in the FL+GM-CSF samples was also significantly higher than the mean yield of DCs in the G-CSF samples. The FL+G-CSF and FL+GM-CSF mobilized DCs were immature by morphologic and phenotypic criteria but stimulated allogeneic T-cells at levels similar to DCs generated in culture from PBMCs. Overnight culture?of the immature DCs obtained from patients receiving either FL+G-CSF or FL+GM-CSF in TNF-alpha?resulted in the generation of mature DCs. In summary, administration of FL in combination with GM-CSF and G-CSF to patients with breast cancer can mobilize large numbers of immature DCs into PBSC leukapheresis collections.

In vitro megakaryocyte expansion in patients with delayed platelet engraftment after autologous stem cell transplantation

Increasing the number of megakaryocytic cells in stem cell transplants by ex vivo expansion culture may provide an approach to accelerate platelet engraftment after high-dose chemotherapy. However, it is unknown if a relationship exists between the expansion potential of progenitor cells and the time to platelet engraftment in vivo. Therefore, we questioned if those patients who potentially would benefit most from expanded cell supplements are able to generate megakaryocytic cells efficiently in vitro. The in vitro megakaryocyte proliferation was analyzed from 19 leukapheresis samples from a group of multiple myeloma patients who all showed rapid neutrophil engraftment, but varied from 7 to 115 days post-transplant to achieve platelet levels >20x10(9)/l. CD34+ cells were isolated and analyzed for their potential to form megakaryocytic colonies (CFU-Mk) in colony assays and megakaryocytic (CD61+) cells in suspension cultures. The frequency and size of CFU-Mk and the expansion potential of CD61+ cells varied eightfold between individual patients. A similar range was found with CD34+ cells isolated from normal bone marrow (n=9). Rapid platelet engraftment occurred in patients receiving both high or low CFU-Mk doses and with high and low expansion of CD61+ cells. Four patients who experienced prolonged (>3 weeks) thrombocytopenia received low CFU-Mk doses, but the expansion potential was around median values or higher. Therefore, we conclude that the megakaryocyte proliferation is not impaired and that in vitro expansion could increase the number of megakaryocytic cells, although other factors could be more relevant in platelet engraftment in this group of patients.