Immunomagnetic Cell Sorting Research Articles

Interleukin‐10‐secreting T cells define a suppressive subset within the HIV‐1‐specific T‐cell population

Recent studies have indicated that Treg contribute to the HIV type 1 (HIV-1)-related immune pathogenesis. However, it is not clear whether T cells with suppressive properties reside within the HIV-1-specific T-cell population. Here, PBMC from HIV-1-infected individuals were stimulated with a 15-mer Gag peptide pool, and HIV-1-specific T cells were enriched by virtue of their secretion of IL-10 or IFN-gamma using immunomagnetic cell-sorting. Neither the IL-10-secreting cells nor the IFN-gamma-secreting cells expressed the Treg marker FOXP3, yet the IL-10-secreting cells potently suppressed anti-CD3/CD28-induced CD4(+) as well as CD8(+) T-cell proliferative responses. As shown by intracellular cytokine staining, IL-10- and IFN-gamma-producing T cells represent distinct subsets of the HIV-1-specific T cells. Our data collectively suggest that functionally defined HIV-1-specific T-cell subsets harbor potent immunoregulatory properties that may contribute to HIV-1-associated T-cell dysfunction.

Activation requirements and responses to TLR ligands in human CD4+ T cells: Comparison of two T cell isolation techniques

Direct regulation of T cell function by microbial ligands through Toll-like receptors (TLR) is an emerging area of T cell biology. Currently either immunomagnetic cell sorting (IMACS) or fluorescence-activated cell sorting (FACS), are utilized to isolate T-cell subsets for such studies. However, it is unknown to what extent differences in T cell purity between these isolation techniques influence T cell functional assays. We compared the purity, response to mitogen, activation requirements, and response to TLR ligands between human CD4+ T cells isolated either by IMACS (IMACS-CD4+) or by IMACS followed by FACS (IMACS/FACS-CD4+). As expected, IMACS-CD4+ were less pure than IMACS/FACS-CD4+ (92.5%±1.4% versus 99.7%±0.2%, respectively). Consequently, IMACS-CD4+ proliferated and produced cytokines in response to mitogen alone and had lower activation requirements compared to IMACS/FACS-CD4+. In addition IMACS-CD4+ but not IMACS/FACS-CD4+ responses were upregulated by the TLR-4 ligand lipopolysaccharide (LPS). On the other hand, TLR-2 and TLR-5 engagement induced costimulation in both IMACS-CD4+ and highly purified IMACS-/FACS-CD4+. Altogether these results indicate that small differences in cell purity can significantly alter T cell responses to TLR ligands. This study stresses the importance of a stringent purification method when investigating the role of microbial ligands in T cell function.

Autoantibody Production during Chronic Graft-Versus-Host Disease Does Not Associate with Long-Term Persistence of Host B Cells in Humans

Background. Chronic graft-versus-host disease (cGvHD) is a common complication of allogeneic hemopoietic cell transplantation (allo-HCT). The pathogenesis of cGvHD is poorly understood. In cGvHD, the homeostasis of B lymphocytes is perturbed, as demonstrated by the production of autoantibodies. B-cell depletion with monoclonal antibodies (mAb) interferes with autoantibody production and ameliorates signs and symptoms of cGvHD. In mouse models, cGvHD and autoantibodies associate with the long-term persistence of host B cells after allo-HCT (Sylvain Perruche et al., Transplantation 2006). It has been postulated that host B cells may present alloantigens to donor T cells and, in turn, receive help for autoantibody production. This could be crucial to the pathogenesis of cGvHD. Aim. To investigate whether the long-term persistence of host B lymphocytes is associated with cGvHD and autoantibodies in humans.Patients and methods. We recruited 13 consecutive patients with active cGvHD (4 mild, 5 moderate, 4 severe according to NIH classification) with a median time of onset of 6 months (range 3–36) from HLA-identical sibling (9 patients) and HLA-matched unrelated (4) allo-HCT. As controls, we chose 10 patients that underwent HLAidentical sibling (2), HLA-matched unrelated (5) or haploidentical (3) allo-HCT and never experienced cGvHD. In the two groups, we studied:circulating autoantibodies, including anti-nuclear (ANA), anti-DNA, anti-extractable nuclear antigen, anti-beta2 glycoprotein, anti-neutrophil cytoplasm, anti-thyroid, anti-mytocondria antibodies, rheumatoid factor,absolute numbers of T (CD3+, CD4+, CD8+), conventional B (CD19+), B1 (CD5+/CD19+) and NK cells (CD16+/CD56+) in the graft and in the peripheral blood,microchimerism by short-tandem repeats (STR) on B, T and myeloid cells purified by immunomagnetic cell sorting (sensitivity 0,01%).Results. Patients with cGvHD had high-titer circulating ANA (>1:160) more frequently than controls (54% versus 10%, P<0,05). All other autoantibodies were negative. Peripheral T-cell counts were lower in patients with cGvHD than in controls (for CD8+ cells P<0,05). This was not due to a difference in the absolute numbers of T lymphocytes within the graft between the two groups. Peripheral counts of conventional B and B1 cells in patients with cGvHD were similar to controls. Autoantibodies and cGvHD were not associated with the persistence of host B lymphocytes, since the analysis of STR on purified B cells revealed that they were all of donor origin. T and myeloid cells were also of donor origin. Of interest, in univariate analysis, in vivo B-cell depletion with mAb for the prophylaxis against Epstein-Barr virus-related lymphoproliferative disease showed a trend towards a lower risk of cGvHD (P=0,06).Conclusions. This study indicates that autoantibody production during cGvHD does not associate with long-term persistence of host B cells in humans. Moreover, it suggests that the early depletion of donor B lymphocytes in vivo may be effective for GvHD prophylaxis

High Level Production of a Lysosomal Enzyme from HSC-Derived Erythroid Cells for Therapeutic Correction of Hurler Syndrome in Mice.

Proviral integration into hematopoietic stem cells (HSC) by lentivirus vector (LV)- mediated gene transfer can provide the benefit of life-long therapeutic effect, yet it can also bring the risk of insertional oncogenesis. Restricting transgene expression to late erythroblasts and red blood cells (RBC) may reduce the risk of activating oncogenes in HSC and its offspring in all lineages. In this study, we sought to evaluate the feasibility of redirecting a fraction of robust protein-synthesis machinery in maturing erythroid cells for production of alpha-L-iduronidase (IDUA), the lysosomal enzyme missing in Mucopolysaccharidosis type I (MPS I, or Hurler Syndrome). We first utilized a murine erythroid leukemia (MEL) cell line to compare IDUA expression and release from LVs using human elongation factor 1α promoter (EF), LTR of SFFV (SF), or a erythroid specific hybrid promoter (IHK) containing human ALAS2 intron 8 erythroid specific enhancer, HS40 core element from human alpha LCR and human ankyrin-1 promoter. We have previously shown highly erythroid-specific expression in vivo using this hybrid promoter, which is sustained in secondary transplantation recipient mice and resists proviral silencing (Moreau-Gaudry et al. Blood, 2001; Mohamedali et al, Mol Ther 2004). MEL cells were introduced to differentiation with hexamethylene bis-acetamide, and progressive erythroid differentiation was confirmed by morphologic evaluation in cytospins. Relatively low levels of expression from IHK (5% of SF, and 8% of EF) were observed in non-induced MEL cells; however, it increased steadily during induction and reached a similarly high expression level as those from the SF promoter. Expression from EF promoter reduced significantly, and the levels from SF promoter remained unchanged with erythroid differentiation. A similar pattern was found in IDUA activities released from stably transduced MEL cells during induction. We then evaluated in vivo the systemic IDUA production in an enzyme-deficient MPS I mouse model using erythroid-specific LV. Lineage-negative bone marrow cells (Lin−) were isolated with 92–97% purity by lineage depletion using immunomagnetic cell sorting. After a short pre-stimulation period, Lin− cells were transduced twice with LV-IHK-IDUA-ires-GFP for a total multiplicity of infection (MOI) at 2–20, resulting in up to 71% transduction efficiency. Four-months after transplantation of Lin- cells transduced at high MOI, sustained and higher-than normal plasma IDUA levels were achieved in all treated MPS I mice (1.5 to 8-fold of normal levels) with GFP transgene expression detected in up to 7% of Ter119+ blood cells. To evaluate transgene expression pattern during erythroid differentiation in bone marrow, we defined erythroblast subpopulations by immunostaining with CD71 and Ter119 for enrichment in proerythroblasts (I), basophilic erythroblasts (II), polychromatophilic erythroblasts (III), and orthrochromatic erythroblasts and reticulocytes (IV). GFP expression was predominantly detected in population III (9–17.3%), followed by the most differentiated population IV. To determine transduction efficiency in HSC, spleen colony-forming-cell assay was performed that showed 15–25% GFP+ spleen colonies in 2o BMT recipients. Moreover, long-term systemic metabolic correction was demonstrated by normalized urinary glycosaminoglycan accumulation in all treated MPS I mice. Complete normalization of tissue pathology was observed in liver and spleen, with relatively moderate improvement in brain. These results demonstrate for the first time that a lysosomal enzyme can be produced and secreted successfully and steadily at superphysiological levels in circulation by erythroid cells using tissue-specific LV, with phenotypic correction of Hurler Syndrome in mice. This data warrants further evaluation for the potential application of erythroid-specific vectors in treating non-RBC related diseases.

Efficient differentiation of hESCs into endothelial cells in vitro is secured by epigenetic changes

Human embryonic stem cells (hESCs) are to be considered as a valuable source for regenerative medicine because of their capacity to differentiate into all cell types. We have developed an efficient culture system to differentiate hECSs into endothelial cells without the formation of embryoid bodies Establishing appropriate culture conditions with a cocktail of growth factors allowed us to differentiate hESCs directly to endothelial primary culture with about 50% efficiency. CD31 immunomagnetic cell sorting was used to purify derived endothelium from the primary culture of hESCs. Isolated endothelial cells expressed immunological markers (vWF, CD105), specific genes (VE-cadherin, KDR, GATA-2, GATA-3, eNOS), and formed cord-like structures on collagen matrix and in Matrigel assay. During differentiation to endothelial lineage promoter regions of the genes involved in specific cell fate determination and homeostasis (GATA-2,-3, and eNOS) underwent intensive hypomethylation which correlated with the gene expression. Overall our data demonstrate that direct differentiation of hESCs leads to endothelial cells that acquire epigenetic patterning similar to the functional endothelial cells of the organism.

Visualization of Circulating Melanoma Cells in Peripheral Blood of Patients with Primary Uveal Melanoma

In patients with uveal melanoma, tumor cell dissemination and subsequent formation of metastases are confined mainly to the hematogenous route. Here, we sought to isolate circulating melanoma cells in peripheral blood of patients with primary uveal melanoma and clinically localized disease. Blood samples from 52 patients with clinically localized uveal melanoma and from 20 control individuals were prospectively collected before therapy of the primary tumor. Tumor cells expressing the melanoma-associated chondroitin sulfate proteoglycan were enriched by immunomagnetic cell sorting and visualized by immunocytologic staining. Results were compared with clinical data at presentation. In 10 of 52 patients [19%; 95% confidence interval (95% CI), 10-33%], between 1 and 5 circulating melanoma cells were detected in 50 mL peripheral blood. No melanoma-associated chondroitin sulfate proteoglycan-positive cells were detected in any of the 20 controls examined. The presence of tumor cells in peripheral blood was associated with ciliary body invasion [odds ratio (OR), 20.0; 95% CI, 3.0-131.7], advanced local tumor stage (OR, 6.7; 95% CI, 1.8-25.4), and anterior tumor localization (OR, 4.0; 95% CI, 1.2-12.7), all established factors for uveal melanoma progression. Immunomagnetic enrichment enables detection of intact melanoma cells in peripheral blood of patients with clinically localized ocular disease. Visualization and capturing of these cells provide a unique tool for characterizing potentially metastasizing tumor cells from a primary melanoma at an early stage of the disease.

Cell Surface Expression of Stem Cell Antigen-1 (Sca-1) Distinguishes Osteo-, Chondro-, and Adipoprogenitors in Fetal Mouse Calvaria

The flat bones of the skull (calvaria) develop by balanced cell proliferation and differentiation in the calvarial sutures and the bone tips. As the brain grows and the calvaria expand, cells within the sutures must remain undifferentiated to maintain suture patency, but osteoprogenitors also need to be recruited into the osteogenic fronts. The exact identity of calvarial osteoprogenitors is currently not known. We used immunomagnetic cell sorting to isolate Sca-1+ and Sca-1(-) cells from fetal mouse calvaria and determined their differentiation potential in in vitro differentiation asssays and in vivo subcutaneous transplantations. Cells within the Sca-1+ cell fraction have a higher adipogenic potential, whereas cells within the Sca-1(-) cell fraction have a higher osteogenic and chondrogenic potential. The Sca-1(-) fraction retains its chondrogenic potential after in vitro expansion but not its osteogenic potential. The Sca-1+ fraction does not retain its adipogenic potential after in vitro expansion. Subcutaneous transplantation resulted in islands of bone and cartilage in implants that had been seeded with Sca-1(-) cells. In conclusion, immunomagnetic cell sorting with Sca-1 antibodies can be used to separate a Sca-1+ cell fraction with adipogenic potential from a Sca-1(-) cell fraction with osteogenic and chondrogenic potential. Isolation of pure populations of calvarial adipoprogenitors, osteoprogenitors, and chondroprogenitors will be beneficial for cellular studies of calvarial development, adipogenesis, osteogenesis, and chondrogenesis. Calvaria-derived osteogenic cell populations may be useful in craniofacial tissue regeneration and repair.

Performance Assessment and Improvement of a Spilt-and-Recombine Micromixer for Immunomagnetic Cell Sorting

Performance of a split-and-recombine micromixer (Tan et al., 2005) is assessed by means of numerical simulation. The micromixer is designed for micro cell sorting systems using immunomagnetic beads, of which working principle is lamination of fluid layers. The fluid motion in a complex three-dimensional geometry is simulated by using the finite difference method and the motion of beads is dealt with the one-way coupling Lagrangian particle tracking method. Although the thickness of laminated layers should ideally reduce by the factor of 2 n (where n is the number of unit-mixers connected in series), the present simulation with a realistic geometry shows that the thickness does not reduce that much. Relatively large unmixed regions are formed in the central region, which deteriorate the mixing performance. The size of the unmixed regions and the degree of mixing are quantitatively investigated. Based on the observation, an improved mixer arrangement is proposed. While the original arrangement proposed by Tan et al. (2005) was to simply connect identical unit-mixers, the present arrangement is to alternately connect the original unit-mixer and one with the mirrored geometry. With this modified arrangement, the unmixed region is suppressed and better mixing is achieved.

Two Classes of Progenitor Cells in Patients with Myeloproliferative Disorders Are Capable of Generating JAK2V617F+CD31+CD144+ Endothelial Cells.

Patients with myeloproliferative disorders (MPD) are at a high risk of developing thrombotic events. We hypothesize that one of the contributory factors to this thrombotic tendency is the involvement of vascular endothelial cells (EC) by the malignant process. In vitro and in vivo assays were used to determine the involvement of EC in patients with MPD. Endothelial progenitor cells (EPC) were assayed from the peripheral blood (PB) mononuclear cells (MNCs) of 3 normal controls (NC) and 16 patients with MPD (12 polycythemia vera (PV), 4 primary myelofibrosis (PMF). MNC were cultured for 2 days in EC growth media on fibronectin(FN)-coated plates. The non-adherent cells were then harvested and transferred to a secondary FN-coated plate for additional 5–14 days. EC colonies were identified by their morphological appearance. The colonies were plucked and analyzed for PECAM-1(CD31), VE-Cadherin(CD144), VEGFR-2, vWF, Endoglin(CD105), ULEX-1, CD45, CD14 by flow cytometry and acetylated LDL(Ac-ADL) uptake. EC colonies were CD31+CD144+VEGFR2+ULEX-1+vWF+CD105+CD45+CD14+ and capable of taking up Ac-LDL and when exposed to TNF-α and IL-1β, expressing ICAM(CD54) and E-selectin(CD62e). MPD MNC formed fewer numbers of EC colonies than normal MNC (31.1±34.2 vs 78.8±28.9; p<0.01) and required more prolonged periods of culture (14 vs 5days). MPD EC colonies were also analyzed for JAK2V617F(JAK2VF) by nested-PCR. 74.6% of MPD EC colonies were homozygous(homo) JAK2VF, 14.9% were heterozygous(hetero) JAK2VF and 10.4% were wild type(wt) JAK2. Interestingly, MNCs from JAK2VF−MPD(148±47) formed greater numbers of EC colonies than NC MNC (78.8±28.9; p≤0.01). MNC from patients with a high burden of JAK2VF alleles (10.3±18.5; p<0.01) formed fewer EC colonies than NC or patients with a low burden of JAK2VF (65.9±28.15; p≤0.01). These EPC assayed in vitro which produced cells with both myeloid and endothelial markers are likely due either to contamination with JAK2VF myeloid cells or the result of the transdifferentiation of myeloid progenitor cells into EC (Bailey A, et al. PNAS. 2006, 103:13156). The inverse correlation between the JAK2VF burden and the ability of MPD MNC to form EC colonies is possibly a consequence of the increased sensitivity of EC to apoptosis due to the constitutive activation of JAK2 (Neria F, et al. Am J Physiol Cell Physiol. 2007, 292:1123). In order to assay for more primitive EPC, 2 cord blood, and 16 JAK2VF+ MPD CD34+ (10 PV, 6 PMF) cells were transplanted into sublethally irradiated NOD/SCID mice. After 8 weeks, EC-rich organs (heart, lung, liver, vessels) were harvested, single cell suspensions were positively selected for either human(h) CD31+or hCD144+ cells by immunomagnetic cell sorting and analyzed for hVEGFR2, CD144, vWF, CD45, CD14 mRNA expression and JAK2VF. These CD31+or CD144+ cells contained transcripts for CD144, vWF, VEGFR2 but not CD45 and CD14. In 77.7% of the cases the hCD31+ or hCD144+ cells were homo JAK2VF, 5.5% were hetero JAK2VF and 16.6% were wt JAK2 and these CD31+or CD144+ cells composed ≤1% of the cells within the respective tissues. hCD144+ cells were also cultured with EC growth media for 7 days and displayed EC morphology and were shown to contain JAK2VF+ cells. These CD31+CD144+JAK2VF+CD14−CD45−cells likely represent the progeny of a malignant EPC which is distinct from an HSC. The involvement of EC by the malignant process in MPD might contribute to the development of thrombosis in MPD.

FISH-Analyses of Cirulating CD34+ Cells in MDS-Patients - A Suitable Method To Measure and Predict Response to 5-Azacytidine.

In this study we present a novel method to measure response to treatment (here 5-Azacytidine (5-Aza)) that is less invasive and can be more frequently performed than bone marrow biopsy (bmb). Usually chromosomal aberrations are proven in bone marrow cells by classical cytogenetics. Most of them are suitable for a detection by FISH-techniques. In our study we have followed chromosomal anomalies in circulating CD34+ cells from peripheral blood in patients (pts) with MDS treated with 5-Aza. We could observe that the size of an abnormal clone in general is larger in circulating CD34+ cells as compared to non-enriched bone marrow cells. The percentage of circulating CD34+ cells showing the respective chromosomal anomaly is decreasing in response to therapy and therefore can be used as an indicator for response. Fourteen pts receiving 5-Aza are included in our ongoing study as yet. Thirteen are male, 1 is female, the average age at the time of diagnosis is 67 years (49–81 years). Starting 5-Aza 7 pts were diagnosed as AML, 3 as RAEB-2, 1 as RAEB-1, 1 as RCMD, 1 as CMML, 1 as RA. In all pts initial karyotyping of bone marrow cells was performed. There was 1 pt with normal karyotype, 5 pts (35.7%) showed a complex aberrant karyotype. The other pts had 1–2 chromosomal anomalies (+8, −7/7q-, 20q-, 5q-). All pts with clonal anomalies (13/14) were informative for further FISH-analyses. In average pts received 4 cycles 5-Aza (range 2–8 cycles). Before every therapy cycle circulating CD34+ cells from peripheral blood were enriched by immunomagnetic cell sorting (MACS) and analysed by FISH. Modified IWG-criteria were used to evaluate hematologic response. At present 10 pts could be analysed completely. All of them showed hematologic and/or cytogenetic response to therapy: 4 pts achieved complete remission (CR), 2 of them after allogeneic bone marrow transplantation. Remarkably, 3 out of 4 evaluable pts with complex aberrant karyotypes responded to 5-Aza. One further pt reached partial remission (PR), one stayed in stable disease (SD). Two pts showed at least a marrow response (PR/CR) without developing other improvements. Two further pts achieved at least hematologic improvement (HI). Remarkably, 9 out of 10 pts showed chromosomal aberrations suitable for FISH. Seven (77.8%) developed partial cytogenetic remission (cyPR) measured in circulating CD34+ cells. Six out of 7 pts with cyPR reached HI and/or CR. One was lost for follw up. CyPR preceded hematologic response as well as progression by 4–8 weeks. We conclude that 5-Aza leads to good results even in high-risk MDS-pts with complex aberrant karyotypes. Our data thus imply that analysing circulating CD34+ cells from peripheral blood by FISH techniques is a suitable method to monitor and to predict response to therapy with 5-Aza and possibly other agents in MDS-pts. It might be the more sensitive method to measure the aberrant cell clone than analysing non-enriched bone marrow cells. It could preserve pts from repeated bmb and allows a very frequent monitoring of therapy response.

CD133 Immunomagnetic Separation: How Effective Is the Method for CD133+ Cell Isolation from Umbilical Cord Blood?.

Umbilical cord blood (UCB) is a rich source of stem cells. Characterisation of stem cell subpopulations residing in UCB requires specific stem cell markers and reliable and reproducible protocols. We assessed the feasibility of CD133 positive cells' isolation using immunomagnetic cell sorting protocols. The separation was based on the manufacturer's instructions (Myltenyi Biotec). We compared these results with the corresponding ones of CD34 isolation. Two additional protocols were also applied to increase the efficiency of the CD133+ yield. The first one included an extra-labeling of the selected CD133 subpopulation from the first column with CD133 microbeads before their application to the second column. The second protocol was an indirect labelling procedure, using CD133-PE monoclonal antibody and either anti-PE microbeads or goat anti-mouse IgG microbeads as well as a combination of them. The CD133 isolation was performed in 18 UCB samples, while the CD34 in 17 samples. The volume of the samples used for CD133+ isolation was 38.5 ± 2.1ml and the respective one for the CD34+ protocol was 40.4 ± 2.8ml. The mononuclear cell fraction had 1.29 ± 0.16 x108 cells and 0.53 ± 0.06% of them expressed CD133. The corresponding values for samples used for CD34 isolation were 2.8 ± 0.3 x108 mononuclear cells and 1.64 ± 0.15% CD34. Following the manufacturer's instructions for CD133 isolation, the number of cells obtained was 21.33 ± 3.3x104 (median 18 x104) and CD133 expression ranged from 10–85% (median=60%). Concerning the CD34 isolation, the number of cells obtained was 54.3 ± 2.1x104 and 93 ± 1.25% expressed CD34. Applying the extra-labeling method, the number of cells obtained was 8±4 x104 and these cells were CD133+ at a percentage of 32.2±4.1%. Applying the indirect immunomagnetic separation the results were as follow: using anti-PE microbeads only, 3 ± 3 x104 cells were retrieved and the percentage of CD133+ was 13.8±9.7. With the use of goat anti-mouse IgG microbeads the cell number obtained was 13±1 x104 of which CD133+ were just 2.0± 2%. In the combination method, the cells initially isolated with anti-PE microbeads and subsequently with goat anti-mouse IgG microbeads were 20 x 104, of which only 18.5% were CD133+. Finally, labeling the cells initially with anti-PE microbeads and consecutively with goat anti-mouse IgG microbeads followed by column isolation yielded 16x104 cells and 19.1% CD133 positive. In conclusion CD34 isolation by the immunomagnetic method results in highly pure CD34+ population confirming the reliability and reproducibility of the method. On the other hand, with the currently available CD133 isolation kit, efficient and reproducible yield of pure CD133 positive population is not feasible.

Rapid Detection and Isolation of Viable Virus-Reactive CD4+ and CD8+ T Cells Based on Activation-Induced CD137 Expression.

Current methods for the detection and isolation of antigen-specific CD4+ and CD8+ T cells require the availability of peptide/MHC multimers or are restricted to cells that produce cytokines after antigen contact. We have recently reported that de novo cell surface expression of the TNF receptor family member CD137 (4-1BB) identifies currently activated, but not resting, human alloreactive CD8+ T cells. This observation allowed us to develop a CD137-based technology for the depletion of alloreactive CD8+ T cells in vitro (Wehler et al. Blood 2007; 109:365–373). More recently, a similar approach has been described that uses activation-induced CD137 expression for the detection and enrichment of antigen-specific CD8+ T cells (Wolfl et al. Blood 2007; 110:201–210). In the current study we complement this work and demonstrate the transient up-regulation of CD137 directly on activated cytomegalovirus (CMV) or Epstein-Barr virus specific CD8+ T cells using peptide/HLA tetramer staining of PBMC from seropositive healthy individuals. Antigen-triggered CD137 expression was first detectable upon 6h of stimulation, and reached peak intensity at 24h, allowing the determination of a clear-cut population of CD137+ T cells at this time point. Most importantly, we also observed a similar CD137 expression kinetics (i.e. low baseline, maximum at 24h) on virus-specific CD4+ T cells upon activation with CMVpp65 peptides. The median frequencies of CMVpp65-reactive CD137+ cells measured ex vivo in 4 different CMV+ healthy donors after 24h of stimulation were 3.6% (range, 1.1–6.7) in CD8+ T cells and 2.7% (range, 0.8–6.4) in CD4+ T cells, respectively. We also analyzed PBMC derived from the same donors and left unstimulated, as well as PBMC from CMV-seronegative donors (n=3) stimulated with CMV peptides. None of these samples contained more than 0.3% CD137+ cells per total CD4+ and CD8+ T cells, thereby confirming the specificity of antigen-induced CD137 expression. We next established a two-step in vitro approach allowing the activation and subsequent CD137-based immunomagnetic cell sorting of virus-reactive CD4+ and CD8+ T cells at the same time. We demonstrated the suitability of this assay to isolate CMVpp65-reactive CD4+ and CD8+ T cells from PBMC of 6 CMV+ healthy individuals. Enriched fractions had a median purity of CD137+ cells of 69.4% (range 12.7–94.1) among CD4+ T cells and 70.6% (range, 28.5–93.4) among CD8+ T cells, respectively. The CD137+ populations could be expanded in vitro and showed CMVpp65-specific cytokine production by CD4+ and CD8+ T cells as well as a strong enrichment of CMVpp65/HLA tetramer-binding CD8+ T cells. We finally compared the efficiency of the CD137 assay with the IFN-γ secretion assay to isolate CMVpp65-specific CD4+ and CD8+ T cells from PBMC. Although both methods were performed at optimal conditions, the numbers of CD137+ T cells measured before and after enrichment were approximately 2-fold higher than those of IFN-γ+ cells (n=5), suggesting that CD137 might detect a broader repertoire of virus-reactive T cells. In conclusion, activation-induced CD137 expression provides a means for the rapid detection and isolation of viable virus-reactive CD4+ and CD8+ T cells. The CD137 assay is most attractive for the simultaneous targeting of both T-cell subsets in monitoring studies and adoptive immunotherapy trials.

Abstract 3354: Increase of Telomere Gap between Granulocytes and Lymphocytes in Patients with Previous Myocardial Infarction: A Novel Measure for Accelerated Telomere Erosion

Individuals with age-corrected reduction of total leukocyte telomere length have been shown to exhibit a remarkable increase in mortality from cardiovascular and infectious complications. Given the considerable interindividual variability of telomere length in healthy adults due to genetic differences already present at birth and the heterogeneity in composition of the total leukocyte compartment, extensive study populations are required to detect accelerated telomere shortening between age-matched groups. With this study, we intended to identify a parameter to improve prediction of individual telomere erosion in patients (pts) with coronary artery disease and previous myocardial infarction (CAD). Methods: Mean telomere length (mTL) was measured in 63 CAD pts (mean age 65±4 years, mean ejection fraction 31±9%) and 23 age-matched healthy controls (65±4 years). Using the flow-FISH method, we were able to distinguish between myeloid and lymphoid cell populations in peripheral blood cells as well as in bone marrow mononuclear cells (BMC). Results: The difference between mTL of the granulocyte and lymphocyte population (ΔTEL Gr-Ly ) was significantly increased from 742 ± 396 base pairs (bp) in the control group to 1055±570 bp in CAD pts (p=0.018). Age was the only independent predictor for telomere length of BMCs. Lymphocytes, but not granulocytes, demonstrated significant telomere shortening between BMCs and peripheral blood in CAD patients (−444 ± 624 bp) as opposed to an increase in a young and healthy control group (+307 ± 372 bp) (p&lt;0.001). Using immunomagnetic cell sorting, telomerase activity (TA) was determined in freshly isolated white blood cell subpopulations. Surprisingly, TA could not be detected in peripheral blood granulocytes (CD15 + granulocytes) from CAD pts. In contrast, CD4 + T-lymphocytes contained the highest amount of TA, compared to CD8 + cells and B-lymphocytes. Conclusions: Our results suggest that primarily peripheral blood lymphocytes - and not granulocytes - can develop stress-induced telomere erosion in CAD. Therefore the base pair length difference between the two populations provides a valuable parameter to detect telomere erosion in patients with CAD, largely independent of the hereditary influence on mTL.

A novel high throughput immunomagnetic cell sorting system for potential clinical scale depletion of T cells for allogeneic stem cell transplantation

To develop an immunomagnetic cell separation system for allogeneic hematopoietic stem cell (HSC) transplantations, which can achieve a high level of T-cell depletion (at least 4.0 log(10)), high level of recovery of hematopoietic stem cells (>90%), with a high throughput (>10(6) cells/second). Peripheral blood leukocytes (PBLs) from buffy coats were spiked with CD34-expressing cells (KG1a) to mimic a leukaphoresis product containing stimulated HSCs. T cells were labeled with anti-CD3(+) Dynabeads and separated in a quadrupole magnetic cell sorter (QMS). The performance of the system with respect to T-cell depletion and recovery of non-T cells and spiked KG1a was determined using four-color, flow cytometry analysis, with the aid of Trucount cell-concentration calibration beads. Limiting dilution assays were also performed to quantify the log(10) depletion of clonable T cells. While the general performance of the QMS system is governed by proven theoretical principles, significant system variability exist, not all of which can be explained by our current understanding. Consequently, a factorial design was employed, guided by JMP software, to optimize the labeling conditions and operation of the QMS focused on maximizing the depletion of T cell, and recovery of unlabeled cells including KG1a cells. From these studies, an optimized, no wash, immunomagnetic labeling protocol and optimized QMS operating conditions were developed. For an average initial cell concentration of 1.7 x 10(8) total cells, an average 3.96 +/- 0.33 log(10) depletion (range, 3.53-4.34) of CD3(+)CD45(+) cells with a mean 99.43% +/- 4.23% recovery of CD34(+)CD45(+) cells (range, 94.38-104.90%) was achieved at a sorting speed of 10(6) cells/s (n = 6). Limiting dilution assays on the T-cell depleted fractions, which gave a log(10) depletion of 3.51 for the clonable T cells. We suggest that this system will provide superior performance with respect to T-cell depletion and CD34(+) recovery for clinical allogeneic bone marrow transplants. Ongoing studies, on a clinical scale, are being conducted to demonstrate this claim.

Isolation of Hematopoietic Stem Cells from Human Cord Blood

Enrichment of hematopoietic stem cells is based on the expression of certain surface antigens, such as CD34 and CD133, or on the lack of expression of lineage-specific antigens. Immunomagnetic positive selection of CD34(+) or CD133(+) cells is performed using paramagnetic microbeads conjugated to specific monoclonal antibodies (anti-human CD34 or anti-human CD133). In negative selection of lineage-negative (Lin(-)) cells, the unwanted cells are labeled with antibodies against known markers for mature hematopoietic cells (CD2, CD3, CD14, CD16, CD19, CD24, CD56, CD66b, and glycophorin A) and retained in the column. Unlabeled cells pass through the column and are collected as the Lin(-) cell fraction. Immunomagnetic cell sorting system MACS is a fast and gentle method to enrich hematopoietic stem cells. Viable and highly pure cells can be separated to be used in various downstream applications, such as flow cytometry and cell culture.

A hepatitis C virus (HCV) NS3/4A protease-dependent strategy for the identification and purification of HCV-infected cells

As a tool for the identification and/or purification of hepatitis C virus (HCV)-infected cells, a chimeric form of the Gal4VP16 transcription factor was engineered to be activated only in the presence of the HCV NS3/4A protease and to induce different reporter genes [choramphenical acetyltransferase (CAT), green fluorescent protein (GFP) and the cell-surface marker H-2K(k)] through the (Gal4)(5)-E1b promoter. For this, the NS5A/5B trans-cleavage motif of HCV of genotype 1a was inserted between Gal4VP16 and the N terminus of the endoplasmic reticulum (ER)-resident protein PERK, and it was demonstrated that it could be cleaved specifically by NS3/4A. Accordingly, transient transfection in tetracycline-inducible UHCV-11 cells expressing the HCV polyprotein of genotype 1a revealed the migration of the Gal4VP16 moiety of the chimera from the ER to the nucleus upon HCV expression. Activation of the chimera provoked specific gene induction, as shown by CAT assay, first in UHCV-11 cells and then in Huh-7 cells expressing an HCV replicon of genotype 1b (Huh-7 Rep). In addition, the GFP reporter gene allowed rapid fluorescence monitoring of HCV expression in the Huh-7 Rep cells. Finally, the chimera was introduced into Huh-7.5 cells infected with cell culture-generated HCV JFH1 (genotype 2a), allowing the purification of the HCV-infected cells by immunomagnetic cell sorting using H-2K(k) as gene reporter. In conclusion, the Gal4VP16 chimera activation system can be used for the rapid identification and purification of HCV-infected cells.

Optimization of immunomagnetic separation for cord blood-derived hematopoietic stem cells

BackgroundThere is a growing interest in cord blood as a source of primitive stem cells with the capacity for multilineage differentiation. Pure cell fractions are needed for the characterization and in vitro expansion of stem cells as well as for their use in preclinical research. However, enrichment of stem cells is challenging due to the lack of stem cell-specific markers and gentle protocols for the isolation of highly pure stem cell fractions. Protocols developed for the enrichment of peripheral blood-derived stem cells have been found to be suboptimal for cord blood.ResultsIn this study, immunomagnetic cell sorting protocols to purify CD34+, CD133+ and Lin- cells from fresh and cryopreserved cord blood were optimized. Reproducible purities of up to 97% were reached. The selected cells were highly viable having substantial colony-forming potential.ConclusionThe optimized protocols enable rapid enrichment of highly pure hematopoietic stem cells from both fresh and cryopreserved cord blood.

Breast carcinoma specific antibody selection combining phage display and immunomagnetic cell sorting

To discover new specific antibodies directed against disseminated carcinoma cells in breast cancer patients, a strategy combining single-chain variable fragment (scFv) phage display and immunomagnetic cell sorting was developed. A selection model, in which ErbB2-expressing breast carcinoma SKBR3 cells are spiked into a 50-fold excess of lymphocytes, was setup. Selection conditions, optimized using the previously characterized ErbB2-specific F5 phage scFv, led to an outstanding phage enrichment yield of 25,000 after only one round. This protocol applied to human naı¨ve and synthetic phage display antibody libraries led to the selection, in only two rounds, of individual scFv clones (43 out of 46 tested) specific for non-epithelial carcinoma antigens expressed on SKBR3 cells. This strategy is fully applicable to metastatic cells in effusions from breast carcinoma patients and shall lead to the discovery of immunotools crucial for novel diagnostic and therapeutic approaches.

Angiotensin II type 1 receptor expression on human leukocyte subsets: A flow cytometric and RT-PCR study

The renin–angiotensin system plays a key role in the regulation of cardiovascular functions and in particular angiotensin II type 1 receptor (AT 1R)-operated pathways are involved in the modulation of inflammation in the vascular wall. In the present study we assessed the pattern of expression of AT 1Rs on different human circulating leukocyte subsets. Venous blood was obtained from healthy male subjects. Leukocyte subsets were purified by immunomagnetic cell sorting or identified in whole blood using multiparametric cytometric analysis. RT-PCR analysis showed that AT 1R mRNA was expressed in polymorphonuclear leukocytes (PMNs), monocytes, B-lymphocytes, and, to a lesser extent, T-lymphocytes. Flow cytometric analysis revealed that the frequency of expression of AT 1Rs was: PMNs > monocytes ≥ B-lymphocytes >> T-lymphocytes, while receptor density per positive cells was: PMNs ≥ B-lymphocytes > T-lymphocytes ≥ monocytes. AT 1Rs are expressed on PMNs, monocytes, T- and B-lymphocytes, however the expression pattern is peculiar to each subset, possibly suggesting distinct roles in the various cell types. Investigating the expression and the functional role of AT 1Rs on circulating leukocyte subsets, as well as their possible modifications in disease conditions before and after pharmacological treatments, is likely to provide novel clues to the comprehension of the mechanisms involved in the therapeutic efficacy of currently available agents.

Filter Buffy Coats (FBC): A source of peripheral blood leukocytes recovered from leukocyte depletion filters

In compliance with federal regulations, blood banks routinely use leukocyte depletion filters to eliminate contaminating leukocytes from blood products such as red blood cell and platelet concentrates. We developed and optimized conditions to elute leukocytes adsorbed to these filters; resulting in leukocyte suspensions which we termed Filter Buffy Coats (FBCs). These Filter Buffy Coats can replace standard buffy coats for various research applications. After optimizing both the filter elution medium as well as elution protocols, we compared commonly used leukocyte depletion filters from four different manufacturers. Relative fractions as well as total recoveries of leukocyte subsets, such as lymphocytes, monocytes and granulocytes, found in Filter Buffy Coats were identified and compared among the filters as well as to standard buffy coats and whole blood. Flow cytometric analysis of Filter Buffy Coats confirmed the presence of T- and B-lymphocytes, NK cells and monocytes. Furthermore, a significant quantity of CD34 + hematopoietic stem or progenitor cells (HSC/HPC) was detected in Filter Buffy Coats prepared from different filters, thus making FBCs a valuable source for research on HSC/HPC. Colony assays revealed that most of these CD34 + cells are functional. Using immunomagnetic cell sorting (MACS), we isolated a variety of leukocyte populations from FBC mononuclear cells (Filter-PBMCs) including T lymphocytes (CD4 +, CD8 +, CD3 +), B lymphocytes (CD19 +), NK cells (CD56 +), HSC/HPC (CD34 +, CD133 +) or dendritic cells (BDCA-4 +). Functional properties of Filter-PBMCs, as well as of some of these isolated leukocyte populations, were confirmed using standard assays. In summary, Filter Buffy Coats are a valuable and convenient source of different peripheral leukocyte populations and can replace standard buffy coat preparations for research applications.