Wir möchten Aaron Selya für hilfreiche Diskussionen danken. Diese Arbeit wurde vom NIH AI29575, CA108835, AI077097 zu JS, ein Pilot Zuschuss von der Johns Hopkins Malaria Research Institute und dem Department of Defense zu gewähren PC 040972 bis MO unterstützt

1. Making, HLA-A2-Ig Basis AAPC <ol><li> Bereiten Sie sterile Boratpuffer <ol><li> Lösen Sie Borsäure in Wasser bis 0,1 zu machen. Den pH-Wert auf 7,0. </li><li> Filter durch einen 0,22 &amp;mgr; m Sterilfilter und lagern bei 4 ° C. </li></ol></li><li> Bereiten Sie sterile Bead Waschpuffer <ol><li> Nehmen Sie 956ml PBS w / o Magnesium oder Calcium. </li><li> Fügen Sie 30ml humanem AB-Serum. </li><li> Add EDTA 2 mM Endkonzentration. </li><li> Add 0.1gsodium Azid. </li><li> Filter durch ein 0,22 um sterile Filter und lagern bei 4 ° C. </li></ol></li><li> Nehmen Sie 1 ml Invitrogen M-450 Epoxy Perlen ab Lager, rund 400 Millionen Beads (Perlen können von Zählkammer gezählt werden), und in eine sterile, Schraubverschluss Glasfläschchen. </li><li> Legen Sie die Flasche gegen eine Dynal Magneten MPC-1, während die Perlen an der Seite des Fläschchens haften, entfernen Sie die Überstände durch Absaugen. Wash Perlen einmal mit 1 ml Boratpuffer. </li><li> Resuspendieren Perlen in einer Mischung aus 1 ml Boratpuffer und 20 ug HLA-A2-Ig-Dimer und 20 ug anti-human CD28 mAb (Clone 9,3). </li><li> Protein zu Wulst Konjugation: put der Glasflasche auf Rotator und drehen bei 4 ° C für 24 Stunden. </li><li> Das Röhrchen wird in MPC-1 Magnet und entfernen Sie alle Boratpuffer. </li><li> Waschen Sie die Kugeln zweimal mit 1 ml Bead Waschpuffer. </li><li> Inkubieren Sie die Perlen in 1ml Bead Waschpuffer bei 4 ° zu drehen C für 24 Stunden. Da Bead Waschpuffer enthält humanem AB-Serum, wird es alle Restprotein Bindungsstelle auf die Perlen zu blockieren. </li><li> Entfernen Sie den Überstand aus Glasflasche, mit 1 ml frisches Bead Waschpuffer zu ersetzen. </li></ol> 2. Qualitätskontrolle von AAPC und Peptid-Beladung, Lagerung <ol><li> Fügen Sie ~ 5 × 10 5 Perlen auf 100 &amp;mgr; FACS-Waschpuffer in FACS-Röhrchen und Fleck mit 1μl der Anti-Maus-IgG1-PE (erkennt den Fc-Teil des HLA-A2-Ig) und 1μl der Anti-Maus-IgG2a-FITC (zu erkennen den Fc-Teil des anti-CD28 mAb). Nach der Färbung für 20 Minuten in FACS-Waschpuffer, wieder waschen und lesen Sie das Färbeergebnis sofort Durchflußzytometer (Abbildung 4). </li><li> Peptide Verladung auf die Perlen: Waschen Sie die Kugeln zweimal in Glasfläschchen mit 1ml sterile PBS. Resuspendieren mit 1 ml sterilem PBS fügen Sie dann 10 &amp;mgr; l von CMV-Peptid (1mg/ml) </li><li> Zählen Sie die Perlen durch Hämozytometer und Etikett der Flasche mit Datum und Konzentration. </li><li> AAPC sind bereit für den Einsatz nach mindestens 3 Tage Peptid Inkubation bei 4 ° C, um genügend Zeit für die Bindung des Peptids auf das HLA-A2-Ig-Dimer zu ermöglichen. Die Perlen können bei 4 ° C gelagert werden und bleiben für mindestens 6 Monate funktionsfähig. </li></ol> 3. Menschliche CTL Isolation <ol><li> Sammeln ~ 100ml frischer peripheren Blut von gesunden HLA-A2 positiven Spender in 10 BD Sodium Heparin Vacutainer-Röhrchen. Verwenden Sie eine 21-Gauge-Nadel oder größer, um eine Hämolyse zu vermeiden. </li><li> Zentrifuge bei 300xg für 10 Minuten bei Raumtemperatur </li><li> Entfernen Sie vorsichtig die obere Plasmaschicht durch Aspiration. Das Plasma kann als Ergänzung für das Kulturmedium verwendet werden. </li><li> Ersetzen Sie das gesammelte Plasma mit sterilem PBS und Transfer das Blut in einem sterilen T75 Kulturflasche oder 50ml konische Röhrchen. Mix das Blut mit PBS gut durch Auf-und Abpipettieren. </li><li> Nach all dem Blut gesammelt werden, bereiten vier weitere 50ml konische Röhrchen und fügen 15ml Ficoll-Paque Plus. </li><li> Langsam Overlay 30-35ml von Blutzellen auf der Oberseite des Ficoll jeder Röhre. Halten Sie die Schnittstelle zwischen den verschiedenen Ficoll und Blutzellen. </li><li> Zentrifugation bei 500 xg für 20 Minuten bei Raumtemperatur. Schalten Sie die Bremse &quot;off&quot; und die Beschleunigung so gering wie möglich eine klare Schnittstelle zwischen den Schichten zu erhalten. </li><li> Mit einem seriological Pipette vorsichtig absaugen PBMC-Schicht und sammeln Sie die PBMC in ein frisches 50ml konischen Rohr. Wenn alle PBMC geerntet add 30 ml PBS und Spin bei 400xg für 10 min. Überstand verwerfen und waschen noch einmal mit 30 ml PBS, um alle verbleibenden Ficoll entfernen. </li><li> Fahren Sie mit CD8 + T-Zell-Isolierung durch Miltenyi menschlichen CD8 + T-Zell-Isolation Kit nach Protokoll des Herstellers. Dieses Kit sehr bereichert für CD8 +-Zellen (in der Regel&gt; 95%) durch Minderung der CD8 - Zellen. </li><li> Zählen Sie die CD8 + T-Zellen. Die erwartete Reinheit sollte größer sein als 95%. Zu diesem Einsatz 2x10 5 Zellen bestätigen und führen Sie eine CD4/3/8 FACS-Analyse. Die restlichen CTL kann entweder sofort für Antigen-spezifische AAPC Stimulation oder sie können für die zukünftige Verwendung eingefroren werden. </li></ol> 4. In vitro AAPC Basis Kultursystem <ol><li> Bereiten Sie Kulturmediums <ol><li> Für TF (T-Zell-Wachstumsfaktor, im Labor 4 hergestellt) 2X Kulturmedium: komplettem RPMI-Medium plus 5% Spender autologem Plasma und 8% T-Zell-Wachstumsfaktor. </li><li&gt; Donor autologem Plasma kann durch Hitze-inaktiviertem humanem AB-Serum ersetzt werden. </li></ol></li><li> Resuspendieren 1 Million CTL in 8ml von TF 2X Kultur-Medium plus 8ml von kompletten RPMI-Medium, fügen Sie 1 × 10 6 AAPC, gut mischen. </li><li> Verwenden Sie ein Multi-Channel-Pipette auf den Teller Zellen auf 96 well U unten Zellkulturplatten. (160 ul pro Well) </li><li> Kultur-Zellen in 37 ° C, 5% CO 2 incubater für 7 Tage. Feed the Zellen an Tag 4 mit 80 ul / well TF 2X Medium. </li><li> Die Zellen sind bereit, am Tag 7 geerntet werden. Nach der Ernte statt die Zellen vor dem Magneten und entfernen Sie die alte AAPC. </li><li> Antigen-Spezifität kann durch Tetramer-Färbung nach dem Protokoll des Herstellers bestimmt werden. Phänotyp Färbung und intrazelluläre Zytokin-Färbung sind nach unserer vorherigen Studie 3 durchgeführt. </li><li> Geernteten Zellen können mit AAPC replattiert wieder unter den gleichen Bedingungen. Zellzahl und Antigen-Spezifität wird erwartet, dass nach wiederholter Stimulation erhöhen. </li></ol> 5. Repräsentative Ergebnisse: Ein Beispiel für AAPC nach HLA A2-Ig und anti-CD28-Konjugation ist in Abbildung 4 dargestellt. Erfolgreiche Proteinkonjugation ist offensichtlich durch eine deutliche Verschiebung der entsprechenden Antikörper-Färbung. Während die Häufigkeit von CMV-spezifischen CTL im peripheren Blut ist in der Regel 0,5-1%, nach einer Woche von AAPC-vermittelte Stimulation kann die Spezifität zu erreichen 55 bis 93% (Abb. 2 und 3). Die Expansion von Antigen-spezifischen CTL kann sehr variabel zwischen verschiedenen Gebern, aber die Ergebnisse sind reproduzierbar innerhalb des gleichen Spenders. Durch Extrapolation kann der Ausbau der CMV spezifische Zellen Tausende von fache verglichen mit Vorstufe Ebenen direkt ex vivo (Daten nicht gezeigt). Intrazelluläre Zytokin-Färbung (Abbildung 3) zeigt, dass diese erweiterte CTL polyfunktionellen, anstatt erschöpft sind, nach längerer Zellkultur und erhebliche Verbreitung. <img src="/files/ftp_upload/2801/2801fig1.jpg" alt="Abbildung 1"/> Abbildung 1. Representative Flussdiagramm AAPC basierte ex vivo Expansion der menschlichen CTL zur adoptiven Immuntherapie bei allogener HSCT <img src="/files/ftp_upload/2801/2801fig2.jpg" alt="Abbildung 2"/> Abbildung 2. Representative Tetramer Färbung durch CMV-spezifischen CTL durch AAPC nach einer Woche der Kultur erzeugt <img src="/files/ftp_upload/2801/2801fig3.jpg" alt="Abbildung 3"/> Abbildung 3. Representative intrazelluläre Zytokin-Färbung aufgrund der CMV-spezifischen CTL durch AAPC generiert (CMV Spezifität betrug 61%) <img src="/files/ftp_upload/2801/2801fig4.jpg" alt="Abbildung 4"/> Abbildung 4. Representative Färbeergebnis der M-450 Epoxy Perlen nach Proteinkonjugation mit Anti-Maus-IgG1-PE und anti-Maus IgG2-FITC gefärbt

<ol>
	<li>Walter, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+cellular+immunity+against+cytomegalovirus+in+recipients+of+allogeneic+bone+marrow+by+transfer+of+T-cell+clones+from+the+donor.">Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T-cell clones from the donor.</a> N Engl J Med. 333, 1038-1038 (1995).</li><li>Rosenberg, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adoptive+cell+transfer:+a+clinical+path+to+effective+cancer+immunotherapy.">Adoptive cell transfer: a clinical path to effective cancer immunotherapy.</a> Nat Rev Cancer. 8, 3669-3669 (2008).</li><li>Oelke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+induction+and+expansion+of+antigen-specific+cytotoxic+T+cells+by+HLA-Ig-coated+artificial+antigen-presenting+cells.">Ex vivo induction and expansion of antigen-specific cytotoxic T cells by HLA-Ig-coated artificial antigen-presenting cells.</a> Nat Med. 9, 619-619 (2003).</li><li>Oelke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+antigen-presenting+cells:+artificial+solution+for+real+diseases.">Artificial antigen-presenting cells: artificial solution for real diseases.</a> Trends Mol Med. 11, 412-412 (2005).</li></ol>

Die AAPC System, das wir hier beschreiben, ist ein effizientes System für die ex vivo Expansion der menschlichen CTL gegen eine Vielzahl von Antigenen. Besondere Vorsicht ist im Hinblick auf die Qualität des Proteins Konjugation und die gleichmäßige Verteilung der AAPC und CTL in der 96-Well-Platte Kultur genommen werden. Mit diesem Ansatz konnten wir CTL erweitern seit mehr als 8 Wochen, in denen wir von Antigen-spezifischen CTL erweitert bis zu einer Million fach 4. Es gab verschiedene künstliche APC-Systeme, die Zell-Linien oder andere azellulären Plattformen 5 worden, aber nach den veröffentlichten Daten jedes System hat sein einzigartiges Profil in Bezug auf Ausbau und Spezifität Unterstützung verschiedener Anwendungen. Wichtig ist, da die Qualität der CTL ist ebenso wichtig wie Quantität, sind die Polyfunktionalität der CMV-spezifischen CTL durch unser System generiert erwartet überlegene antivirale Wirksamkeit zu verleihen.

<table border="1"><tbody><tr><th> Reagens </th><th> Firma </th><th> Katalog-Nummer </th></tr><tr><td> Vacutainer-Röhrchen (enthält Heparin) </td><td> Becton Dickinson </td><td> 367874 </td></tr><tr><td> Menschliche CD8 + T-Zell-Isolation Kit </td><td> Miltenyi </td><td> 130-094-156 </td></tr><tr><td> Dynabeads M-450 Epoxy </td><td> Invitrogen </td><td> 140,11 </td></tr><tr><td> Dynal MPC-1 Magnet </td><td> Invitrogen </td><td> 120-01D </td></tr><tr><td> Ficoll-Paque Plus- </td><td> GE Healthcare </td><td> 17-1440-03 </td></tr><tr><td> RPMI Medium 1640 </td><td> Gibco </td><td> 11875 </td></tr><tr><td> HLA-A2-Ig-Dimer X </td><td> Becton Dickinson </td><td> 551263 </td></tr><tr><td> iTAgMHC Tetramer (HLA-A2-CMV)-PE </td><td> Beckman Coulter </td><td> T20099 </td></tr><tr><td> Falcon klare 96-well Mikrotiterplatte </td><td> Becton Dickinson </td><td> 353077 </td></tr><tr><td> Rat anti-Maus-IgG2a-FITC </td><td> Becton Dickinson </td><td> 553390 </td></tr><tr><td> Ziege anti-Maus-IgG1-PE </td><td> Invitrogen </td><td> P21129 </td></tr><tr><td> Humanem Serum-Typ AB </td><td> Atlanta Biologicals </td><td> S40110 </td></tr><tr><td> Maus anti-human CD8a-FITC </td><td> Sigma-Aldrich </td><td> F0772 </td></tr><tr><td> Maus anti-human CD8a-APC </td><td> Becton Dickinson </td><td> 340684 </td></tr><tr><td> Maus anti-human IFN &amp;ggr;-FITC </td><td> Becton Dickinson </td><td> 340449 </td></tr><tr><td> Maus anti-human TNF-PE </td><td> Becton Dickinson </td><td> 340512 </td></tr></tbody></table>

hla-ig based artificial antigen presenting cells for efficient ex vivo expansion of human ctl

CTL mit optimaler Effektor-Funktion spielen eine entscheidende Rolle bei der Vermittlung von Schutz gegen verschiedene intrazelluläre Infektionen und Krebs. Jedoch können Einzelpersonen weisen suppressive Immunsystem Mikroumgebung und im Gegensatz zu aktivierenden CTL, die autologe Antigen-präsentierende Zellen können dazu neigen, tolerize oder anergize Antigen-spezifischen CTL. Als Ergebnis, obwohl noch in der experimentellen Phase, hat CTL-basierte adoptiven Immuntherapie entwickelt, um eine viel versprechende Behandlung für verschiedene Krankheiten wie Krebs und Virusinfektionen werden. In ersten Experimenten ex vivo expandierten CMV (Cytomegalovirus) spezifische CTL sind für die Behandlung von CMV-Infektion bei immunsupprimierten allogenen Knochenmark-transplantierten Patienten eingesetzt. Zwar ist es üblich, lebensbedrohliche CMV-Virämie bei diesen Patienten haben, keiner der Patienten erweitert CTL entwickeln CMV Erkrankung, was bedeutet, das Anti-CMV Immunität von der adoptiv übertragen CTL 1 hergestellt. Vielversprechende Ergebnisse wurden auch für Melanome beobachtet worden und kann auf andere Arten von Krebs 2 verlängert werden. Zwar gibt es viele Möglichkeiten, um ex vivo zu stimulieren und zu erweitern menschlichen CTL sind, werden aktuelle Ansätze der Kosten und technische Grenzen beschränkt. Zum Beispiel ist der derzeitige Goldstandard für die Verwendung von autologen DC. Dies erfordert jeden Patienten zu einer signifikanten Anzahl der Leukozyten zu spenden und ist auch sehr teuer und aufwändig. Darüber hinaus hat detaillierte In-vitro-Charakterisierung der DC erweitert CTL ergab, dass diese nur suboptimal Effektor-Funktion 3 haben. Hier präsentieren wir eine hocheffiziente AAPC basiertes System für die ex vivo Expansion von humanen CMV-spezifischen CTL zur adoptiven Immuntherapie (Abbildung 1). Die AAPC wurden durch Kopplung Zelle magnetische Kügelchen mit humanen HLA-A2-Ig-Dimer-und Anti-CD28mAb 4. Sobald AAPC gemacht sind, können sie mit verschiedenen Peptiden von Interesse geladen werden und bleiben für Monate funktionsfähig. In diesem Bericht wurden AAPC mit einer dominanten Peptid aus CMV, pp65 (NLVPMVATV) geladen. Nach der Kultivierung humaner CD8 + CTL von einem gesunden Spender mit AAPC für eine Woche, kann CMV-spezifischen CTL dramatisch in die Spezifität erhöht werden bis zu 98% (Abbildung 2) und verstärkt mehr als 10.000-fach. Wenn mehr CMV-spezifische CTL erforderlich sind, kann die weitere Expansion leicht durch repetitive Stimulation mit AAPC erreicht werden. Phänotypische und funktionelle Charakterisierung zeigt diese expandierten Zellen haben ein Effektor-Memory-Phänotyp und machen erhebliche Mengen der beiden TNF und IFN &amp;ggr; (Abbildung 3).

Watch this Scientific Journal Video about HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL at JoVE.com

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

Ein neuer DC unabhängige Methode zur Induktion und Expansion von Antigen-spezifischen T-Zellen beschrieben. HLA A2-Ig Basis künstlicher Antigen-präsentierende Zellen (AAPC) sind mit HLA-A2 restringierte Peptide effizient erweitern CTL verschiedener Antigen-Spezifität geladen. Diese Technologie birgt ein großes Potenzial für die CTL-basierte adoptiven Immuntherapie.

HLA-Ig Basierend Artificial Antigen-präsentierende Zellen für eine effiziente Ex vivo Erweiterung des menschlichen CTL

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals ...

hla-ig-based-artificial-antigen-presenting-cells-for-efficient-ex

Research

JoVE Journal

Immunology and Infection

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

15.3K Aufrufe.  Johns Hopkins University. Ein neuer DC unabhängige Methode zur Induktion und Expansion von Antigen-spezifischen T-Zellen beschrieben. HLA A2-Ig Basis künstlicher Antigen-präsentierende Zellen (AAPC) sind mit HLA-A2 restringierte Peptide effizient erweitern CTL verschiedener Antigen-Spezifität geladen. Diese Technologie birgt ein großes Potenzial für die CTL-basierte adoptiven Immuntherapie.

Serie: HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation. This labeling has classically been used to study proliferation and migration. In the method presented here, we have shortened the timeline after adoptive transfer to look at survival and killing of epitope specific CFSE labeled target cells4-6. The level of specific killing of a CD8 + T cell clone can indicate the quality of the response, as their quantity may be misleading. Specific CD8+ T cells can become functionally exhausted over time with a decline in cytokine production and killing7,8. Also, certain CD8 + T cell clones may not kill as well as others with differing TCR specificities9. For effective Cell Mediated Immunity (CMI), antigens must be identified that produce not only adequate numbers of responding T cells, but also functionally robust responding T cells. Here we assess the percent cell specific killing of two peptide specific T cell clones in BALB/c mice.

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Many infections elicit a strong CTL response, but occasionally, the quantity of responding cells does not correlate to control of the pathogen1. One measure of CTL quality is their ability to kill specifically2. CFSE labeling of target cells can be used to investigate this CTL response quality in vivo3,4.

Antigen-spezifische In Vivo Killing-Assay mit CFSE markierten Zielzellen

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation.  ...

Forschung

Immunologie und Infektion

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide complete protection against the development or recurrence of breast cancer. To overcome this problem by increasing the frequency of tumor-reactive T cells, adoptive immunotherapy has been employed. A variety of protocols have been used for the expansion of tumor-specific T cells. These protocols, however, are restricted to the use of tumor antigens ex vivo for the activation of antigen-specific T cells. Very recently, common gamma chain cytokines such as IL-2, IL-7, IL-15, and IL-21 have been used alone or in combination for the enhancement of anti-tumor immune responses3. However, it is not clear what formulation would work best for the expansion of tumor-reactive T cells. Here we present a protocol for the selective activation and expansion of tumor-reactive T cells from the FVBN202 transgenic mouse model of HER-2/neu positive breast carcinoma for use in adoptive T cell therapy of breast cancer. The protocol includes activation of T cells with bryostatin-1/ionomycin (B/I) and IL-2 in the absence of tumor antigens for 16 hours. B/I activation mimics intracellular signals that result in T cell activation by increasing protein kinase C activity and intracellular calcium, respectively4. This protocol specifically activates tumor-specific T cells while killing irrelevant T cells. The B/I-activated T cells are cultured with IL-7 and IL-15 for 24 hours and then pulsed with IL-2. After 24 hours, T cells are washed, split, and cultured with IL-7 + IL-15 for additional 4 days. Tumor-specificity and anti-tumor efficacy of the ex vivo expanded T cells is determined.

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

An efficient protocol for the ex vivo expansion of tumor-reactive T cells from tumor-draining lymph nodes or other secondary lymphoid tissues of tumor-bearing hosts is described. This protocol selectively expands tumor-specific T cells for use in adoptive immunotherapy of breast cancer.

Ex vivo Expansion von Tumor-reaktiven T-Zellen mittels Bryostatin 1/Ionomycin und der Gemeinsamen gamma-Kette Zytokine Formulation

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide ...

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

Expansion, Reinigung und Functional Assessment of Human peripheren Blut NK-Zellen

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

Expansion of Human Peripheral Blood  &amp;#947;&amp;#948; T Cells using Zoledronate

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

Expansion von humanen peripheren Blut γδ T-Zellen mit Zoledronat

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules2. NKT cells express an invariant TCR&alpha; chain rearrangement: V&alpha;14J&alpha;18 in mice and V&alpha;24J&alpha;18 in humans, which is associated with V&beta; chains of limited diversity3-6, and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d 7. The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with &alpha;-Galactosylceramide (&alpha;-GalCer) and a variety of cytokines8. Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines9,10. In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients11. However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells12, 13. Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is a fundamental requirement of NKT cell activation, antigen: CD1d-Ig complexes provide a reliable method to isolate, activate, and expand effector NKT cell populations.

Artificial Antigen Presenting Cell aAPC Mediated Activation and Expansion of Natural Killer T Cells

Here we describe a method for activating and expanding human NKT cells from bulk T cell populations using artificial antigen presenting cells (aAPC). The use of CD1d-based aAPC provides a standardized method for generating high numbers of functional NKT cells.

Künstliche Antigen-präsentierenden Zelle (AAPC) vermittelte Aktivierung und Expansion der Natural Killer-T-Zellen

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike ...

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

Klinische Anwendung der<em&gt; Dornröschen</em&gt; Und künstliche Antigen präsentierenden Zellen genetisch zu verändern T Zellen aus dem peripheren und Nabelschnurblut

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

Beurteilung der Innate Sensing von HIV-1-infizierten CD4<sup&gt; +</sup&gt; T-Zellen durch Plasmazytoide Dendritische Zellen unter Verwendung eines<em&gt; Ex-vivo-</em&gt; Co-Kultursystem.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

Killer Künstliche Antigen-präsentierenden Zellen (KaAPC) für effiziente<em&gt; In-vitro-</em&gt; Depletion von humanen Antigen-spezifischen T-Zellen

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

Lymphocyte Isolierung von menschlicher Haut für phänotypische Analyse und<em&gt; Ex Vivo</em&gt; Zellkultur

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/- recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts.
This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

Entwicklung einer Präsentation von Antigenen zu studieren Antigen-driven ulcerosa Modell durch Antigen-präsentierende Zellen zu T-Zellen präsentieren

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...