detection-and-isolation-of-viable-mouse-il-17-secreting-t-cells

Detection and Isolation of Viable Mouse IL-17-Secreting T Cells

The MACS Cytokine Secretion Assay technology allows detection of secreted cytokines on the single cell level and sensitive isolation of viable ...

T helper (Th) lymphocytes can develop memory for the expression of particular cytokines, like IL-4 or IL-10, in that reexpression of those cytokines is independent of the original costimulatory signal IL-4 and depends only on T cell receptor stimulation. Here, we show that in the course of Th2 cell differentiation in vitro, IL-4 memory is established during primary activation of naÃ¯ve Th cells, whereas the establishment of IL-10 memory requires repetitive stimulation of the Th cell with IL-4 and T cell receptor. Likewise, established IL-10 memory, maintained in the absence of further IL-4 signals, was observed in individual IL-10-producing cells generated from in vivo antigen-experienced CD62L(low) Th cells and isolated by using the newly developed cytometric cytokine secretion assay for IL-10. In naÃ¯ve Th cells undergoing primary activation, the induction of both IL-4 and IL-10 memory requires DNA synthesis, but reexpression of the cytokine genes can occur throughout cell cycle. In in vitro polarized Th2 cell populations, Th cells with IL-4 or IL-10 memory do not differ in proliferative behavior. Populations of Th cells isolated from polarized Th2 cultures according to expression of IL-4 or IL-10 also do not differ in proliferative behavior. Their proliferation mainly depends on IL-2. Thus, effector memory Th lymphocytes with memory for IL-4 or IL-10 expression are not intrinsically impaired in their proliferative potential and can play an essential role in reactive immunological memory and its regulation.

Establishment of memory for IL-10 expression in developing T helper 2 cells requires repetitive IL-4 costimulation and does not impair proliferation.

Gr&#xFC;ndung der Speicher f&#xFC;r IL-10-Expression in sich entwickelnden T-Helfer-2-Zellen erfordert wiederholte IL-4-Kostimulation und beeintr&#xE4;chtigt nicht die Proliferation.

Adoptive transfer of cytomegalovirus (CMV)-specific T cells can restore long-lasting, virus-specific immunity and clear CMV viremia in recipients of allogeneic stem cell transplants if CD4(+) and CD8(+) CMV-specific T cells are detected in the recipient after transfer. Current protocols for generating virus-specific T cells use live virus, require leukapheresis of the donor, and are time consuming. To circumvent these limitations, a clinical-scale protocol was developed to generate CMV-specific T cells by using autologous cellular and serum components derived from a single 500-mL blood draw. CMV-specific T cells were stimulated simultaneously with CMV-specific major histocompatibility complex class I (MHC I)- restricted peptides and CMV antigen. Activated T cells were isolated with the interferon-gamma (IFN-gamma) secretion assay and expanded for 10 days. In 8 randomly selected, CMV-seropositive donors, 1.34 x 10(8) combined CD4(+) and CD8(+) CMV-specific T cells, on average, were generated, as determined by antigen-triggered IFN-gamma production. CMV-infected fibroblasts were efficiently lysed by the generated T cells, and CMV-specific CD4(+) and CD8(+) T cells expanded if they were stimulated with natural processed antigen. On the other hand, CD4(+) and CD8(+) T cell-mediated alloreactivity of generated CMV-specific T-cell lines was reduced compared with that of the starting population. In conclusion, the culture system developed allowed the rapid generation of allodepleted, highly enriched, combined CD4(+) and CD8(+) CMV-specific T cells under conditions mimicking good manufacturing practice.

Rapid generation of combined CMV-specific CD4+ and CD8+ T-cell lines for adoptive transfer into recipients of allogeneic stem cell transplants.

Schnelle Generation von kombinierten CMV-spezifischen CD4 + und CD8 + T-Zell-Linien f&#xFC;r den adoptiven Transfer in Empf&#xE4;ngern allogener Stammzelltransplantation.

Stem cell transplantation is used widely in the management of a range of diseases of the hemopoietic system. Patients are immunosuppressed profoundly in the early posttransplant period, and reactivation of cytomegalovirus (CMV) remains a significant cause of morbidity and mortality. Adoptive transfer of donor-derived CMV-specific CD8+ T cell clones has been shown to reduce the rate of viral reactivation; however, the complexity of this approach severely limits its clinical application. We have purified CMV-specific CD8+ T cells from the blood of stem cell transplant donors using staining with HLA-peptide tetramers followed by selection with magnetic beads. CMV-specific CD8+ cells were infused directly into nine patients within 4 h of selection. Median cell dosage was 8.6 x 10(3)/kg with a purity of 98% of all T cells. CMV-specific CD8+ T cells became detectable in all patients within 10 d of infusion, and TCR clonotype analysis showed persistence of infused cells in two patients studied. CMV viremia was reduced in every case and eight patients cleared the infection, including one patient who had a prolonged history of CMV infection that was refractory to antiviral therapy. This novel approach to adoptive transfer has considerable potential for antigen-specific T cell therapy.

Adoptive transfer of cytomegalovirus-specific CTL to stem cell transplant patients after selection by HLA-peptide tetramers.

Adoptiven Transfer von Cytomegalovirus-spezifischen CTL zu Zelle transplantierten Patienten nach der Auswahl durch HLA-Peptid-Tetramere einzud&#xE4;mmen.

The adoptive transfer of donor CD4+CD25+ regulatory T cells has been shown to protect from lethal graft-versus-host disease after allogeneic bone marrow transplantation in murine disease models. Efficient isolation strategies that comply with good manufacturing practice (GMP) guidelines are prerequisites for the clinical application of human CD4+CD25+ regulatory T cells. Here we describe the isolation of CD4+CD25+ T cells with regulatory function from standard leukapheresis products by using a 2-step magnetic cell-separation protocol performed under GMP conditions. The generated cell products contained on average 49.5% CD4+CD25high T cells that phenotypically and functionally represented natural CD4+CD25+ regulatory T cells and showed a suppressive activity comparable to that of CD4+CD25+ regulatory T-cell preparations purified by non-GMP-approved fluorescence-activated cell sorting.

Isolation of CD4+CD25+ regulatory T cells for clinical trials.

Der adoptive Transfer Spender CD4 + CD25 + regulatorischen T-Zellen wurde zum Schutz vor t&ouml;dliche Graft - Versus - Host Disease nach allogene Knochenmark-Transplantation in murinen Krankheitsmodelle gezeigt. Effiziente Isolierung-Strategien, die gute Herstellungspraxis (GMP) Richtlinien einzuhalten sind Voraussetzungen f&uuml;r die klinische Anwendung der menschlichen CD4 + CD25 + regulatorischen T-Zellen. Hier beschreiben wir die Isolation von CD4 + CD25 + T-Zellen mit hoheitlichen Funktion von Norm Leukapheresis Produkte mit einem magnetischen Zellseparation 2-Schritt-Protokoll unter GMP-Bedingungen durchgef&uuml;hrt. Die erzeugten Produkte enthalten durchschnittlich 49,5 % CD4 + CD25high T-Zellen, die ph&auml;notypisch und funktionell nat&uuml;rliche CD4 + CD25 + regulatorischen T-Zellen vertreten und einer unterdr&uuml;ckende Aktivit&auml;t vergleichbar, zeigte dass von CD4 + CD25 + regulatorische T-Zell-Vorbereitungen durch GMP-nichtzugelassenen Fluoreszenz aktivierte Zellsortierung gereinigt.

Isolierung von CD4 + CD25 + regulatorischen T-Zellen f&uuml;r klinische Studien.

The cell-surface affinity matrix technology, otherwise called "secretion assay" or "capture assay," represents a new, innovative method for the analysis and enrichment of viable cells according to secreted molecules such as antibodies or cytokineof T cells to induce cytokine secretion is included. Alternate protocols provide very rapid and simple procedures for the direct detection of antigen-specific T cells from small numbers of PBMCs or whole blood without magnetic enrichment.

Detection and isolation of cytokine secreting cells using the cytometric cytokine secretion assay.

Die Zelloberfl&auml;che Affinit&auml;t Matrix Technologie, sonst &quot;Sekretion-Assay&quot; oder &quot;Aufnahme-Assay,&quot; genannt stellt eine neue, innovative Methode zur Analyse und Anreicherung von entwicklungsf&auml;higen Zellen nach sezernierte Molek&uuml;le wie Antik&ouml;rper oder Cytokineof T-Zellen zu Zytokin-Sekretion induzieren ist enthalten. Alternative Protokolle bieten sehr schnelle und einfache Verfahren zum direkten Nachweis von Antigen-spezifische T-Zellen von kleinen St&uuml;ckzahlen PBMCs oder Vollblut ohne magnetische Anreicherung.

Article	Year
Establishment of memory for IL-10 expression in developing T helper 2 cells requires repetitive IL-4 costimulation and does not impair proliferation. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 14514890	2003
Rapid generation of combined CMV-specific CD4+ and CD8+ T-cell lines for adoptive transfer into recipients of allogeneic stem cell transplants. Blood\| PubMed ID: 14670917	2004
Adoptive transfer of cytomegalovirus-specific CTL to stem cell transplant patients after selection by HLA-peptide tetramers. The Journal of experimental medicine\| PubMed ID: 16061727	2005
Isolation of CD4+CD25+ regulatory T cells for clinical trials. Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation\| PubMed ID: 16503495	2006
Detection and isolation of cytokine secreting cells using the cytometric cytokine secretion assay. Current protocols in immunology / edited by John E. Coligan ... [et al.]\| PubMed ID: 18432889	2002

Mario Assenmacher

Biography

JoVE Journal Publications

Other Publications