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Results for “T cell selection”

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At least 145 records · Page 8Linked to original sources
PubMed2000

Immunomagnetic tumor cell selection--implications for the detection of disseminated cancer cells.

BACKGROUND: The optimal method for the detection of disseminated epithelial cancer cells has not yet been found. The standard method, using immunocytochemistry, offers a sensitivity of up to 10(-6). Molecular methods such as cytokeratin-19 RT-PCR are about 10 times as sensitive, but they are hampered by interference such as illegitimate gene expression. STUDY DESIGN AND METHODS: Immunomagnetic bead selection of epithelial cancer cells using conjugates directed against the human epithelial antigen (HEA) followed by immunocytochemistry testing was investigated in this trial. RESULTS: No cytokeratin-positive cells could be enriched from 56 control samples. In 104 clinical samples of bone marrow aspirations, PBPC collections, and venous blood obtained from breast cancer patients, the cytokeratin-positive rate increased significantly, from 29.9 percent before selection to 54.8 percent after enrichment. Even the yield of detected cancer cells was significantly higher after selection. Up to 2.5 x 10(8) MNCs were easily processed. However, the mean cancer cell recovery after HEA enrichment was only 24.4 percent. Subsequently, selected epithelial cells were successfully immunophenotyped by use of a double-stain technique detecting cytokeratin-positive cells and the urokinase-like plasminogen activator receptor. CONCLUSION: HEA bead selection in combination with the standard immunocytochemistry method is a powerful and specific tool for the detection of disseminated cancer cells without false-positive results. Furthermore, it delivers enough cells for subsequent investigations such as characterization studies.

Antigens, Surface↗
PubMed1979

Segregation of pre-stalk and pre-spore cells of Dictyostelium discoideum: observations consistent with selective cell cohesion.

Aggregating Dictyostelium discoideum cells were dissociated and separated into two populations, pre-stalk and pre-spore cells, by centrifugation in a Percoll gradient. After harvesting, one population of cells was labeled with fluorescein isothiocyanate and the other with tetramethylrhodamine isothiocyanate. Equal numbers of the two cell populations were then mixed and observed while they aggregated and differentiated to form fruiting bodies. Within minutes after mixing, the two populations showed evidence of segregation and tended to form separate streams of cells as they migrated toward aggregation centers. Many separate clusters of tetramethylrhodamine isothiocyanate-labeled or fluorescein isothiocyanate-labeled cells were found within an aggregation center. Thereafter there was further segregation. The denser cells from the gradient tended to assume the position of stalk cells whereas the lighter cells migrated to the position characteristic of spore cells. The patterns of segregation suggest that selective cohesiveness of one or both cell types plays a role in morphogenesis.

Cell Aggregation↗
PubMed1998

The level of CD4 surface protein influences T cell selection in the thymus.

During T cell development thymocytes are subjected to positive and negative selection criteria to ensure that the mature T cell repertoire is MHC restricted, yet self tolerant at the same time. The CD4 and CD8 coreceptors are thought to play a crucial role in this developmental process. To elucidate the role of CD4 in T cell selection, we have produced a mouse strain that expresses CD4 at a reduced level. We used homologous recombination in embryonic stem cells to insert neo into the 3' untranslated region of CD4. The resulting mice have a reduction in the percentage of CD4+ cells in the thymus and a concomitant increase in CD8+ cells. In addition, breeding two individual class II-restricted TCR transgenic mice onto the CD4low (low level of CD4) mutant background affects the selection of each TCR differentially. In one case (AND TCR transgenic), significantly fewer CD4+ cells with the transgenic TCR develop on the CD4low mutant background, whereas in the other (5C.C7 TCR transgenic), selection to the CD4 lineage is only slightly reduced. These data support the differential avidity model of positive and negative selection. With little or no avidity, the cell succumbs to programmed cell death, low to moderate avidity leads to positive selection, and an avidity above a certain threshold, presumably above one that would lead to autoreactivity in the periphery results in clonal deletion. These data also support the idea that a minimum avidity threshold for selection exists and that CD4 plays a crucial role in determining this avidity.

Animals↗
PubMed1989

Sequential amplification of dihydrofolate reductase and multidrug resistance genes in Chinese hamster ovary cells selected for stepwise resistance to the lipid-soluble antifolate trimetrexate.

We describe the development of resistance to trimetrexate and piritrexim (BW 301U) by a stepwise selection protocol in Chinese hamster ovary cells. Selection in trimetrexate resulted in initial resistance as a result of dihydrofolate reductase gene amplification. Several trimetrexate-resistant variants that display 250-340-fold and 25-50-fold resistance to lipophilic and hydrophilic antifolates, respectively, were established. Increased antifolate resistance was associated with a prominent overexpression of dihydrofolate reductase as determined from the elevated folate reductase activity, cellular labeling with fluorescein-methotrexate, and steady-state mRNA levels as a result of a consistent dihydrofolate reductase gene amplification. However, upon subsequent incremental increases in trimetrexate, further resistance was also associated with amplification of the multidrug resistance gene. This resulted in overexpression of P-glycoprotein and a subsequent 20-50-fold collateral resistance to pleiotropic drugs such as adriamycin, actinomycin D, vinca alkaloids, etoposide, and colchicine. In contrast, initial resistance following selection with low piritrexim concentrations resulted from an unknown mechanism(s) not involving overproduction of either dihydrofolate reductase or P-glycoprotein. This piritrexim resistance was shared with trimetrexate but not with methotrexate. Upon further selection with piritrexim, resistant variants emerge with amplified dihydrofolate reductase but not with multidrug resistance genes. These variants were subsequently resistant to both hydrophilic and lipophilic folate antagonists but retained sensitivity to pleiotropic drugs. The pattern of resistance with methotrexate, trimetrexate, and piritrexim shared a common mechanism, dihydrofolate reductase gene amplification, but differed regarding the additional amplification of the multidrug resistance gene in trimetrexate-resistant cells as well as the emergence of an additional unknown mechanism(s) of resistance to lipid-soluble antifolates upon initial selection in piritrexim.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2006

Rapid analysis of T-cell selection in vivo using T cell-receptor retrogenic mice.

Although T-cell receptor (TCR) transgenic as well as knockout and knockin mice have had a large impact on our understanding of T-cell development, signal transduction and function, the need to cross these mice delays experiments considerably. Here we provide a methodology for the rapid expression of TCRs in mice using 2A peptide-linked multicistronic retroviral vectors to transduce stem cells of any background before adoptive transfer into RAG-1(-/-) mice. For simplicity, we refer to these as retrogenic mice. We demonstrate that these retrogenic mice are comparable to transgenic mice expressing three commonly used TCRs (OT-I, OT-II [corrected] and AND). We also show that retrogenic mice expressing male antigen-specific TCRs (HY, MataHari and Marilyn) facilitated the analysis of positive and negative selection in female and male mice, respectively. We examined various tolerance mechanisms in epitope-coupled TCR retrogenic mice. This powerful resource could expedite the identification of proteins involved in T-cell development and function.

Animals↗
PubMed2000

Germinal center initiation, variable gene region hypermutation, and mutant B cell selection without detectable immune complexes on follicular dendritic cells.

Serum antibody (Ab) can play several roles during B cell immune responses. Among these is to promote the deposition of immune complexes (ICs) on follicular dendritic cells (FDCs). ICs on FDCs are generally thought to be critical for normal germinal center (GC) formation and the development and selection of memory B cells. However, it has been very difficult to test these ideas. To determine directly whether FDC-bound complexes do indeed function in these roles, we have developed a transgenic (Tg) mouse in which all B lymphocytes produce only the membrane-bound form of immunoglobulin M. Immune Tg mice have 10,000-fold less specific Ab than wild-type mice and lack detectable ICs on FDCs. Nonetheless, primary immune responses and the GC reaction in these mice are robust, suggesting that ICs on FDCs do not play critical roles in immune response initiation and GC formation. Moreover, as indicated by the presence and pattern of somatic mutations, memory cell formation and selection appear normal in these IC-deficient GCs.

Amino Acid Sequence↗
PubMed2003

Strategies to reduce transplant-related mortality after allogeneic stem cell transplantation in elderly patients: Comparison of reduced-intensity conditioning and unmanipulated peripheral blood stem cells vs a myeloablative regimen and CD34+ cell selection.

OBJECTIVES: The aim of this study was to compare two approaches used to reduce transplant-related mortality (TRM) after allogeneic peripheral blood stem cell transplantation (allo-PBSCT) in elderly patients. PATIENTS AND METHODS: Data from 50 patients, 45 years of age or older, consecutively treated with an HLA-identical sibling allo-PBSCT at the Hospital de Sant Pau were analyzed. We have compared the outcome of patients treated with conventional myeloablative regimens and CD34(+)-selected cells (CD34(+) group; n=23) with those receiving reduced-intensity conditioning regimens, consisting of fludarabine (150 mg/m(2)) plus an alkylating agent, followed by unmanipulated grafts (RIC group; n=27). Patient characteristics were well balanced between the two groups, although patients in the RIC group were slightly older. RESULTS: The incidence of acute graft-vs-host disease (GVHD) was similar in both groups. The 1-year cumulative incidence of extensive chronic GVHD was 38% in the RIC group and 17% in the CD34(+) group (p=0.2). After a median follow-up of 28 months, there were no differences in the relapse rate. Patients in the RIC group had a lower TRM, with a cumulative incidence of 7% vs 30% at 6 months and 15% vs 39% at 1 year (p=0.05). The Kaplan-Meier estimates of PFS at 2 years was 67% in the RIC group and 43% in the CD34(+) group (p=0.09) and the OS was 69% vs 43% (p=0.05), respectively. CONCLUSION: CD34(+) cell selection reduced the risk of extensive cGVHD but was associated with a higher TRM. Although the number of patients is limited, our study suggests that this approach should be restricted to relatively young patients, as better outcomes can be achieved in elderly patients using RIC strategies.

Aged↗
PubMed1992

Overexpression of P-glycoprotein and alterations in topoisomerase II in P388 mouse leukemia cells selected in vivo for resistance to mitoxantrone.

The overexpression of P-glycoprotein (PGP) and alterations in DNA topoisomerase II (TOPO II) were evaluated in mouse leukemia P388 cells selected in vivo for mitoxantrone (MTT) resistance (P388/MTT) and compared to doxorubicin (DOX) resistant (P388/DOX) or vincristine (VCR) resistant (P388/VCR) models. Among a panel of TOPO II inhibitors which included etoposide (VP-16), DOX, MTT and 4'-[(9-acridinyl)-amino]methanesulfon-m-anisidide (m-AMSA), the relative resistance compared to parental sensitive P388/S cells was: P388/DOX greater than P388/MTT greater than P388/VCR. All the resistant sublines exhibited minimal cell kill (less than 20%) at vincristine concentrations greater than 100-fold the IC50 for P388/S cells. In a soft-agar colony-forming assay, the modulation of cytotoxicity in P388/MTT cells by the calmodulin inhibitor trifluoperazine following a 3-hr drug treatment demonstrated a marked potentiation in cell kill with MTT, VP-16, DOX and m-AMSA but not VCR. Immunoblotting data revealed that while PGP was not detectable in P388/S cells, the overexpression of PGP was apparent in P388/MTT cells and the relative expression between the resistant sublines was: P388/DOX greater than P388/MTT greater than P388/VCR. Although the amount and DNA cleavage activity of TOPO II in nuclear extracts from P388/VCR cells were comparable to those in P388/S cells, they were markedly lower in both P388/DOX and P388/MTT cells. However, decatenation activity of TOPO II in nuclear extracts was comparable between the sensitive (P388/S) and resistant sublines (P388/MTT, P388/DOX, and P388/VCR). Results from the present study demonstrated that P388 cells selected for resistance to mitoxantrone exhibit changes in TOPO II and overexpression of PGP similar to P388/DOX cells, while vincristine resistant cells only overexpress PGP. Since therapeutic strategies are primarily designed to interfere with PGP-mediated drug efflux, the choice of agents for modulating resistance in tumors which overexpress PGP versus tumors which overexpress PGP with altered TOPO II could be different.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

A novel three-pronged approach to kill cancer cells selectively: concomitant viral, double suicide gene, and radiotherapy.

Two obstacles limiting the efficacy of nearly all cancer gene therapy trials are low gene transduction efficiencies and the lack of tumor specificity. Recently, a replication-competent, E1B-attenuated adenovirus (ONYX-015) was developed that could overcome these limitations, because it was capable of efficiently and selectively destroying tumor cells lacking functional p53. In an attempt to improve both the efficacy and safety of this approach, we constructed a similar adenovirus (FGR) containing a cytosine deaminase (CD)/herpes simplex virus type-1 thymidine kinase (HSV-1 TK) fusion gene, thereby allowing for the utilization of double-suicide gene therapy, which has previously been demonstrated to produce significant antitumor effects and potentiate the therapeutic effects of radiation. The FGR virus exhibited the same tumor cell specificity and replication kinetics as the ONYX-015 virus in vitro. Importantly, both the CD/5-FC and HSV-1 TK/GCV suicide gene systems markedly enhanced the tumor cell-specific cytopathic effect of the virus, and, as expected, sensitized tumor cells to radiation. By contrast, neither the FGR virus nor either suicide gene system showed significant toxicity to normal human cells. Both suicide gene systems could be used to suppress viral replication effectively, thereby providing a means to control viral spread. The results support the thesis that the three-pronged approach of viral therapy, suicide gene therapy, and radiotherapy may represent a powerful and safe means of selectively destroying tumor cells in vivo.

Adenoviridae↗
PubMed1997

Abnormal T cell selection on nod thymic epithelium is sufficient to induce autoimmune manifestations in C57BL/6 athymic nude mice.

To investigate the role of primary T cell repertoire selection in the immunopathogenesis of autoimmune diseases, pure thymic epithelium (TE) from nonobese diabetic (NOD) embryos was grafted into non autoimmune prone newborn C57BL/6 athymic mice. The results show that NOD TE selects host T cell repertoires that establish autoimmunity in otherwise nondiabetic animals. Thus, such chimeras regularly show CD4 and CD8 T cell-mediated insulitis and sialitis, in contrast with syngeneic or allogeneic chimeras produced with TE from nonautoimmune strains. This is the first demonstration that autoimmunity to pancreatic beta cells and salivary glands can be established by the sole alteration of the thymic environment involved in T cell selection, regardless of the nature and presentation of both major histocompatibility complex and tissue-specific antigens on the target organ. These data indicate that T cell repertoire selection by the NOD thymic epithelium is sufficient to induce specific autoimmune characteristics in the context of an otherwise normal host.

Animals↗
PubMed1997

Agonist peptide modulates T cell selection thresholds through qualitative and quantitative shifts in CD8 co-receptor expression.

Engagement of the TCR is a pivotal step in thymocyte development, ultimately resulting in the survival (positive selection) or loss (negative selection) of developing T cells. The roles of peptides and stromal cell interactions necessary for these selection events, however, are still poorly understood. To investigate the effects of agonist peptide in positive selection, we used a novel cell suspension model for in vitro thymic positive selection in adults. Target thymocytes from H-2Db-restricted TCR transgenic mice, specific to the lymphocytic choriomeningitis virus (LCMV) peptide bred on a non-selecting MHC background (H-2d or TAP-1-/-), were co-cultured with freshly isolated H-2b thymic stromal cells. In the presence of selecting stroma the nominal agonist LCMV peptide induced apoptosis at high concentrations and at low concentrations enhanced the efficiency of positive selection both in numbers of cells 'rescued' and kinetics of appearance of selected single-positive cells. We further illustrate down-modulation of CD8 alpha beta or CD8 beta at high but non-deleting concentrations of agonist peptide. This highlights the ability of the T cell, within the window of positive selection, to modify surface co-receptors both qualitatively and quantitatively in response to increasing avidity TCR-peptide-MHC interactions. The direct consequence of this would be to lower the total signaling events below the threshold for apoptosis induction. Hence if self peptide were not presented in sufficient quantities in the thymus, autoreactive cells may escape deletion and may actually be positively selected.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2004

Cocultures of metastatic and host immune cells: selective effects of NAMI-A for tumor cells.

The effects of NAMI-A, [H2im][trans-RuCl4(dmso-S)(Him)], a new metal-based agent for treating tumor metastases, have been investigated in vitro on splenocytes, ConA- or LPS-activated T and B lymphoblasts, and thymocytes. Splenocytes and thymocytes exposed for 1 h to 0.01-0.1-mM NAMI-A do not change their mitochondrial functionality, cell cycle distribution, protein synthesis, and CD44 expression in comparison to untreated control samples. Instead, mitochondrial functionality increased 24 h after treatment in a fraction of splenocytes. The same treatment reduced mitochondrial functionality and S phase of the cell cycle in T and B blasts (already after 1 h treatment) and reduced CD44 expression on B blasts, 24 h after treatment. On cocultures of splenocytes and metastatic cells (metGM) (1:1), NAMI-A induces a selective depolarization of mitochondrial membrane potential of metGM cells, while it stimulates splenocytes (mainly lymphocytes), as shown by the increase of the S phase, nitric oxide production, and adhesion onto metastatic cells. This, in turn, reduces the number of metastatic cells and results in the increased ratio between splenocytes and metGM in favor of diploid cells (doubling from one to two). Rosetting of leukocytes onto metastatic cells correlates with induction of CD54 expression on tumor cells after NAMI-A in vivo treatment, which in turn, might contribute to metastasis recognition by cytotoxic lymphocytes. The overall antimetastatic activity displayed by NAMI-A might therefore be the result of complex interactions with tumor cells, on which it displays selective antitumor activity, and with host immune cells through which it promotes activation of host immune defenses involved in tumor suppression.

Animals↗
PubMed1994

Increased Activity of [gamma]-Glutamylcysteine Synthetase in Tomato Cells Selected for Cadmium Tolerance.

Two cell lines of tomato (Lycopersicon esculentum Mill cv VFNT-Cherry) were systematically compared for their capacity to tolerate cadmium. Unselected CdS cells died in the presence of 0.3 mM CdCl2. CdR6-0 cells, which were selected from CdS, survived and grew in medium supplemented with 0.3 mM CdCl2. Growth of CdR6-0 cells under this condition was accompanied by synthesis of cadmium-binding phytochelatins and maintenance of cellular glutathione (GSH) levels. CdR6-0 cells also exhibited increased tolerance to buthionine sulfoximine, in both the presence and absence of 0.1 mM CdCl2. The specific activity of [gamma]-glutamylcysteine synthetase (EC 6.3.2.2) was approximately 2-fold higher in CdR6-0 cells than in CdS cells, whereas there was no difference between cell lines in specific activity of GSH synthetase (EC 6.3.2.3). Increased activity of the first enzyme of GSH biosynthesis in CdR6-0 cells, presumably a result of selection for increased cadmium tolerance, provides an enhanced capacity to synthesize GSH and to maintain the production of phytochelatins in response to cadmium. This adaptation may contribute to the enhanced cadmium tolerance of CdR6-0 cells.

Journal Article↗
PubMed1996

T cell selection and autoimmunity: flexibility and tuning.

Peptide-MHC interactions govern the fate of T cells in the thymus and the peripheral T cell repertoire. Recent progress has involved investigating how different peptides influence T cell selection and mature T cell function and the subsequent implications for tolerance and autoimmunity.

Amino Acid Sequence↗
PubMed2005

Immunomagnetic cell selection performed for HLA haploidentical transplants with the CliniMACS device: effect of additional platelet removal on CD34+ cell recovery.

Immunomagnetic CD34+ cell selection (ICS) is a widely employed technology in autotransplant and allotransplant settings. When an haploidentical transplant is performed, a high dose of purified CD34+ cells together with efficient T and B cell depletion are required to minimize the risks of graft versus host disease (GVHD) and Epstein-Barr virus (EBV)-related lymphoma. To ameliorate the performances of the CliniMACS (Miltenyi Biotec) ICS device, we compared 73 ICS performed following the manufacturer's recommended platelet depletion versus 48 performed after adjunctive centrifugations to increase platelet depletion of the leukapheresis (LKF) product. A total of 121 ICS (from single or fractioned LKF products) were carried out on 93 LKF collected from 47 related healthy donors. A statistical significance in terms of CD34+ cell recovery (81.8% vs. 71.2%) was found in favor of the modified ICS procedure (p=0.0049) with a comparable stem cell purity and viability. The modification of the standard manufacturer's technique for increasing platelet depletion can further improve the recovery of stem cells with no influence on T and B cell depletion. These results demonstrate the negative influence exerted on CD34+ cell recovery by LKF platelet contamination.

Adolescent↗
PubMed1996

Natural killer and lectin-dependent cytotoxic activities of Kurloff cells: target cell selectivity, conjugate formation, and Ca++ dependency.

Kurloff cells may represent a major component of NK cell activity in the guinea pig. We have pursued to characterize the mechanism of their action. Using murine target cells, we found Kurloff cell cytotoxicity to be selective for the NK-sensitive YAC-1 target cell, with minimal activity against the NK-resistant P815 target cell. In the presence of PHA, but not ConA, cytotoxicity was markedly augmented against both YAC-1 and P815. While effector-target conjugate formation was observed with YAC-1 cells but not P815 cells in control cultures, it was augmented with both target cell types in cultures with PHA. Pretreatment alone with PHA was ineffective, however. NK cell activity of Kurloff cells was dependent on extracellular Ca++ and entry of Ca++ into the effector cells, as demonstrated by abrogation of cytotoxicity when extracellular Ca++ was chelated with EDTA or EGTA, or following treatment with the Ca++ channel blockers verapamil and diltiazem. Furthermore, inhibition of PKC by H7 resulted in significant reduction of Kurloff cell-mediated NK activity, while pretreatment of effector cells with the PKC activator TPA enhanced NK activity. Kurloff cells could also be stimulated to produce serine esterases by contact with target cells or treatment with phorbol ester and ionophore. Finally, a majority of Kurloff cells, identified by the monoclonal antibody 14D1, reacted with the human NK cell marker CD56. Taken together, these data suggest that Kurloff cells have NK-like characteristics and activity, with target cell selectivity, and that their lytic mechanisms involve influx of extracellular Ca++, PKC activation and serine esterase production.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗