A new safeguard eliminates T cell receptor gene-modified auto-reactive T cells after adoptive therapy

Dissertation

zur Erlangung des akademischen Grades

doctor rerum naturalium
(Dr. rer. nat.)

im Fachbereich Biologie
eingereicht an der

Mathematisch-Naturwissenschaftlichen Fakultät I
der Humboldt-Universität zu Berlin
von

Diplom - Biologin   Elisa   Kieback
geboren am 01.12.1980 in Dresden

Präsident der Humboldt-Universität zu Berlin
Prof. Dr. Dr. h.c. Christoph Markschies

Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I
Prof. Dr. Lutz-Helmut Schön

Gutachter:
1. Prof. Dr. Wolfgang Uckert
2. Prof. Dr. Thomas Blankenstein
3. Prof. Dr. Hinrich Abken

Tag der mündlichen Prüfung: 20.10.2008

To my parents

Summary

The adoptive transfer of T cell receptor (TCR) gene-modified T lymphocytes into patients is associated with potential risk factors. First, auto-immunity may occur if a tumor-associated antigen is targeted on normal tissue, if TCR chain mispairing leads to the formation of an auto-reactive receptor or if an otherwise anergic endogenous receptor specific for an auto-antigen becomes activated. Second, retroviral integration could lead to malignant transformation of the T cell. Therefore, it is essential to have the possibility to deplete the transferred T cells in vivo in case of severe side effects. The available safety modalities such as suicide gene/prodrug systems or cell surface proteins that are targeted by specific antibodies comprise disadvantages rendering them less feasible for the application in adoptive therapy with TCR gene-modified T cells.

In this thesis, a new safeguard has been developed which is based on a TCR-intrinsic depletion mechanism and can eliminate auto-reactive TCR-redirected T cells. By introducing a 10 amino acid-long sequence of the human c-myc protein (myc-tag) into the murine OT-I and P14 TCRs or the human gp100 TCR it was possible to deplete TCR-expressing T cells in vitro and in vivo with a myc-specific antibody. Depending on the antibody isotype, either complement lysis or antibody-dependent cell-mediated cytotoxicity could be induced. The T cells maintained equal function compared to cells expressing the wild-type receptor as shown by MHC-tetramer binding and cytokine secretion. 

Importantly, the in vivo depletion of adoptively transferred T cells could prevent disease in an auto-immune mouse model. Here, splenocytes transduced with a myc-tagged OT-I TCR were injected into RIP-mOVA mice which express the OT-I-specific antigen ovalbumin in the β-islet cells of the pancreas. Destruction of these cells by the adoptively transferred T cells led to severe diabetes in untreated mice. Animals which received a myc-specific antibody after T cell transfer remained healthy and showed no increase in blood glucose levels.

The developed safeguard allows termination of adoptive therapy in case of severe side-effects. The strategy is superior to previous ones as it relies on a TCR-intrinsic mechanism which does not require introduction of an additional gene. Safety is not hampered by loss or low expression of the transgene and immunogenicity in humans is unlikely.

Zusammenfassung

Der adoptive Transfer von T-Zellrezeptor- (TZR-) modifizierten T Zellen ist mit potentiellen Risiken verbunden. Erstens können Autoimmunreaktionen auftreten, wenn Tumor-assoziierte Antigene auf normalem Gewebe erkannt werden, Fehlpaarung der TZR-Ketten zur Bildung eines autoreaktiven Rezeptors führen oder ein sonst anerger endogener Rezeptor aktiviert wird, der ein Autoantigen erkennt. Zweitens besteht das Risiko der malignen Transformation der Zelle durch Insertionsmutagenese des Retrovirusvektors. Daher ist es notwendig, die transferierten T Zellen im Falle schwerer Nebenwirkungen eliminieren zu können. Verfügbare Sicherheitsmechanismen wie Suizidgene oder Oberflächenmoleküle, die von spezifischen Antikörpern erkannt werden, sind für adoptive Therapie mit TZR-modifizierten T Zellen aufgrund vieler Nachteile ungeeignet.

In dieser Arbeit wurde ein neuer Sicherheitsansatz entwickelt, der auf einem TZR-intrinsischen Depletionsmechanismus beruht und autoreaktive, TZR-veränderte T Zellen eliminieren kann. Durch Einfügen einer 10 Aminosäure-langen Sequenz des humanen c-myc Proteins (myc-tag) in murine (OT-I, P14) und humane (gp100) TZRs konnten TZR-exprimierende T Zellen in vitro und in vivo mittels eines myc-spezifischen Antikörpers depletiert werden. Abhängig vom Isotyp des Antikörpers konnte Komplement-abhängige Lyse oder Antikörper-vermittelte zelluläre Zytotoxizität gezeigt werden. Die T Zellen behielten vergleichbare Funktionalität hinsichtlich Antigenerkennung und Zytokinsekretion wie Zellen, die den Wild-Typ Rezeptor exprimierten.

Die i n vivo Depletion adoptiv transferierter T Zellen verhinderte lethalen Diabetes in einem Mausversuch. Im verwendeten Modell wurden Splenozyten, die mit einem myc-getagten OT-I TZR transduziert wurden, in RIP-mOVA Mäuse injiziert, die in den Inselzellen des Pankreas das OT-I-spezifische Antigen Ovalbumin exprimieren. Zerstörung der Inselzellen durch die transferierten T-Zellen induzierte lethalen Diabetes in unbehandelten Mäusen. Tiere, denen ein myc-spezifischer Antikörper verabreicht wurde, zeigten keine Symptome. Dieser neuartige Sicherheitsmechanismus erlaubt es, adoptive T Zelltherapie abzubrechen, falls schwere Nebenwirkungen auftreten. Im Gegensatz zu früheren Strategien beruht diese auf einem TZR-intrinsischen Mechanismus, bei dem kein zusätzliches Gen eingebaut werden muss. Die Sicherheit des Ansatzes wird durch Verlust oder Herunterregulierung des Transgens nicht beeinflusst und Immunogenität im Menschen ist unwahrscheinlich.

Keywords: T cell receptor, gene therapy, safety, myc-tag

Eigene Schlagworte: T Zellrezeptor, Gentherapie, Sicherheit, myc-tag

Inhaltsverzeichnis

• 1 Introduction
  • 1.1 The T cell receptor complex
  • 1.2 T cell immuno-therapy for cancer
    • 1.2.1 Transfer of unmodified T cells
    • 1.2.2 Transfer of gene-modified T cells
  • 1.3 Risk factors of adoptive T cell therapy
    • 1.3.1 Recognition of tumor-associated antigens on self-tissue
    • 1.3.2 Formation of heterodimers by endogenous and transgenic TCR  
    • 1.3.3 Activation of an endogenous auto-reactive TCR
    • 1.3.4 Insertional mutagenesis and transformation
  • 1.4 Potential safety mechanisms
    • 1.4.1 Suicide gene/prodrug systems
    • 1.4.2 Apoptosis-inducing fusion genes and dimerizer prodrugs
    • 1.4.3 CD20 and CD20-depleting antibodies
    • 1.4.4 Cytotoxic tetramers
  • 1.5 Outline of this thesis
• 2  Material and Methods
  • 2.1 Material
    • 2.1.1 Oligonucleotides
    • 2.1.2 Plasmids and retroviral vectors
    • 2.1.3 Peptides and tetramers
    • 2.1.4 Mice strains
    • 2.1.5 Cell types and media
    • 2.1.6 Antibodies
  • 2.2 Methods
    • 2.2.1 Molecular biology
    • 2.2.2 Cell culture
  • 2.3 Companies
• 3  Results
  • 3.1 Introduction of a myc-tag into different positions of the murine P14 TCR
  • 3.2 Expression analysis of myc-tagged P14 TCRs
  • 3.3 Myc-tagged P14 TCRs allow in vitro cell depletion
  • 3.4 Functionality of myc-tagged P14 TCR is retained
  • 3.5 Generation and characterization of OT-I/TCRmyc – a second murine TCR with a myc-tag
  • 3.6  In vivo depletion of T cells transduced with myc-tagged TCRs
  • 3.7 Depletion of TCRmyc-transduced T cells in a mouse model of auto-immune disease 
  • 3.8 Expression and function of the human myc-tagged TCR gp100
  • 3.9  In vitro depletion of human T cells expressing myc-tagged TCR gp100
• 4  Discussion
  • 4.1 Generation and expression of myc-tagged TCRs
  • 4.2 Depletion of TCRmyc-transduced T cells in vitro
  • 4.3 Function of TCRmyc-transduced T cells
  • 4.4 Depletion of TCRmyc-transduced T cells in vivo
  • 4.5 Advantages of the TCRmyc safeguard over others
  • 4.6 Implementation of the safeguard into a clinical setting
    • 4.6.1 Availability of a myc-specific depleting antibody
    • 4.6.2 Universality of the safeguard for different TCRs
  • 4.7 Eventual limitations of myc-tagged TCRs as a safeguard
    • 4.7.1 Immunogenicity of TCRmyc
    • 4.7.2 Elimination of activated T cells
    • 4.7.3 Elimination of transformed T cells
    • 4.7.4 Elimination of T cells expressing TCR heterodimers
    • 4.7.5 Activation of auto-reactive T cells by the myc-specific antibody
  • 4.8 Future prospect
•  Abbreviations
• References
• Acknowledgements
•  Publications
• Statement

Tabellen

• Table 1 : Safety modalieties for adoptive T cell transfer
• Table 2 : Positions of myc-tag insertion in the murine P14 TCR.

Bilder

• Figure 1 :   Schematic and crystal structure of the αβ   TCR complex. (A) A schematic drawing of a TCR, showing the TCRα-chain (purple) and the TCRβ-chain (blue) linked by a dilsulfide bond (yellow). Accessory CD3 molecules (green) mediate the signal transduction via ITAM motifs (red boxes). (B) Crystal structure of the extra-cellular part of the murine 2C TCR. The numbers in the V regions indicate the CDR loops. Adapted from [6 ]. (C) Presumed model of a TCR-CD3 cluster.
• Figure 2 : HSV-TK can phosphorylate thymidine and nucleotide analogs. (A) Thymidine is phosphorylated by cellular kinases or HSV-TK to thymidine-mono-phosphate (dTMP) and further to thymidine-tri-phosphate (dTTP) which can be incorporated into a replicating DNA strand. To the free OH- group, further nucleotides can be attached. (B) HSV-TK also phosphorylates nucleotide analogs like GCV, which in their tri-phosphorylated form are integrated in the newly synthesized DNA. As no further nucleotides can be attached to GCV, the replication process is discontinued.
• Figure 3 : Mechanism of apoptosis induction by a caspase 9 fusion construct. (A) Pathway of Fas-induced apoptosis. By binding of Fas ligand (FasL) to the Fas receptor (FasR) the receptor molecules form a cluster bringing the death effector domains (DED, red boxes) in close proximity. This recruits FADD which subsequently leads to activation of downstream caspases. Cytochrome C release from mitochondria mediates the conversion of pro-caspase 9 into its activated form. (B) Structure of the caspase 9-FKBP fusion construct. Upon application of the prodrug the molecules multimerize which mimics the process of caspase 9 activation.
• Figure 4 : Design of site-directed mutagenesis PCR using pairs of overlapping primers. Green: Primers specific for the TCR sequence. Red: Myc-tag or P2A sequence.
• Figure 5 : Regions for insertion of a myc-tag in to a TCR.  Crystal structure of the 2C TCRα-chain (purple) and TCRβ-chain (blue). The arrows indicate the four regions selected for insertion of the myc-tag. (1: N-terminus of TCRα-chain, 2: c-strand of Cα, 3: N-terminus of TCRβ-chain, 4: FG loop of Cβ). Adapted from [6, ].
• Figure 6 : Positions of myc-tag insertion in the murine P14 TCR. The 10 aa myc-tag sequence was incorporated into the sequence of the TCRα- (A) or TCRβ-chain (B). In five variants, sequences of the original TCR were exchanged by one or two myc-tag sequences (1). Numbers indicate the first and last aa position of the original sequence that was replaced. Four variants were generated, in which one or two myc-tags were inserted (2). Here, numbers indicate the amino acid position behind which the tag was introduced. The name of each variant according to Table 2 is given in brackets. (S: signal peptide, V: variable region, D: diversity region, J: joining region, C: constant region). The asterix marks the position [DAN] which was later used for modification of different human and murine TCRs.
• Figure 7 :   Fusion of a myc-tag to the N-terminus of the mature P14 TCR α – and TCR β –chain s does not alter the signal peptide cleavage position. (A,B) The peptide sequence of the P14 TCRα– and TCRβ–chain was analyzed using SignalP 3.0 software. The position with the highest Y score indicates the putative cleavage site of the signal peptide (VNG-QQ for the α–chain and MEA-AV for the β–chain). (C,D) Similarly, the modified P14/TCRmyc[AN] and P14/TCRmyc[BN] amino acid sequences which carry a myc-tag between the proposed signal peptide and the mature protein were analyzed. The myc-tag sequence is boxed in red.
• Figure 8 :   All myc-tagged P14 TCR s are expressed in the murine T cell line B3Z. B3Z cells were transduced with one of the P14/TCRmyc and subsequently sorted for vα2 and vβ8 expression. (A) Cells were stained with vα2- and vβ8-specific antibodies and analyzed by flow cytometry. The number of double-positive cells after sorting is given in percentage. Untransduced cells (neg) or cells transduced with P14/TCRwt (both boxed in grey) were used as a control. (B) Mean fluorescence intensity (MFI) of the TCRα-chain (black bars) and TCRβ-chain (grey bars) staining was determined. The depicted relative MFI values indicate the expression level of the myc-tagged TCR relative to expression of the wt TCR chain which was set to 100%.
• Figure 9 : Only some myc-tag modified TCRs can be detected by a myc-specific antibody. (A) P14/TCRmyc-transduced and –enriched B3Z cells were analyzed by FACS using a myc-specific antibody. Untransduced cells (neg) or cells transduced with P14/TCRwt (both boxed in grey) were used as controls. Numbers indicate the percentage of sorted cells that stained positive with the antibody. (B) An overlay of the histograms shows distinct levels of myc-tag detection of different TCRs.
• Figure 10 :   P14/TCRmyc [ AN, DAN, DL and XL ] are expressed in a TCR-deficient T cell line. 58 cells were transduced with P14/TCRmyc [AN, DAN, DL and XL] retroviral vectors and subsequently sorted for vα2 and vβ8 expression. Cells were stained with vα2- and vβ8-specific antibodies and analyzed by flow cytometry. The number of transduced cells after sorting is given in percentage. Untransduced cells (neg) or cells transduced with P14/TCRwt (both boxed in grey) were used as a control. 
• Figure 11 :   P14/TCRmyc [ AN, DAN, DL and XL ]   can be detected by a myc-specific antibody   when ex pressed in a TCR-deficient murine T cell line. (A) P14/TCR-transduced 58 cells that were enriched for TCR expression were analyzed by FACS using a myc-specific antibody. Untransduced cells (neg) or cells transduced with P14/TCRwt (both boxed in grey) were used as controls. Numbers indicate the percentage of sorted cells that stained positive with the antibody. (B) An overlay of the histograms shows distinct levels of myc-tag detection of different TCRs.
• Figure 12 :   P14/TCRmyc[ DAN ] and [ DL ]   support complement d epletion of T cells. (A) 58 cells transduced with P14/TCRmyc [AN, XL, DL or DAN] and enriched for TCR expression were incubated with a myc-specific antibody and rabbit complement factors. Viable and dead cells were discriminated by staining with 7-AAD and FACS analysis. P14/TCRwt-transduced cells were used as a negative control (boxed in grey). Numbers indicate the percentage of dead cells for specific (blue) or unspecific lysis (grey). (B) From these data, percentage of specific depletion was calculated. The experiment was performed twice with reproducible results.
• Figure 13 :   P14/TCRmyc function s comparable to P14/ TCR wt. (A) B6 splenocytes were transduced with P14/TCRwt or P14/TCRmyc. After 72 hours the cells were stained with a CD8-specific antibody, a P14-specific MHC-tetramer and an irrelevant tetramer (irr). Untransduced cells (neg, boxed in grey) served as a negative control. Cells shown are gated on CD8 expression. Numbers indicate the MFI of the specific tetramer staining. The experiment was repeated twice with similar results. (B) For peptide titration, 58 (CD8α+) cells were transduced with P14/TCRwt or P14/TCRmyc. Cells were stained with vα2- and vβ8-specific antibodies and analyzed by FACS. Transduction efficiency is given in percentage. Untransduced 58 (CD8α+) cells were used as a negative control (neg, boxed in grey). (C) 1 x 10⁵ P14/TCRwt- or P14/TCRmyc-transduced or untransduced 58 (CD8α+) cells (neg) were stimulated for 24 hours with 1 x 10⁵ B6 splenocytes pulsed with 100 µM to 100 pM gp33 peptide. IL-2 concentration of the culture supernatant was analyzed by ELISA. Unloaded splenocytes cells (w/o) or splenocytes loaded with irrelevant peptide (irr) served as negative controls. Data represent mean values of duplicates and error bars indicate the standard deviation (SD).
• Figure 14 : Expression of OT-I/TCRmyc and OT-I/TCRwt is comparable. TCR-deficient 58 cells were transduced with OT-I/TCRmyc or OT-I/TCRwt retroviruses and enriched by FACS with a vβ5-specific antibody. TCR expression was detected by staining with vα2- and vβ5-specific antibodies (A) or a myc-specific antibody (B) and anlyzed by flow cytometry. Untransduced 58 cells (neg, boxed in grey) served as a negative control for TCR expression. Numbers indicate the percentage of sorted double-positive T cells.
• Figure 15 : OT-I/TCRmyc- transduced 58 cells can be depleted by complement. (A) TCR-deficient 58 cells were transduced with OT-I/TCRmyc and enriched with vβ5-specific antibodies. For depletion, cells were incubated with rabbit complement factors and a myc-specific antibody (blue) or rabbit complement alone as a control (grey). 7-AAD was used to discriminate between living and dead cells. Percentages represent 7-AAD-positive, dead cells. OT-I/TCRwt-transduced cells served as a negative control (boxed in grey). (B) From these data, percentage of specific depletion was calculated. The results represent data from one of at least two independent experiments with comparable results.
• Figure 16 : Myc-tagged OT-I TCR functions comparable to wt OT-I TCR. (A) B6 splenocytes transduced with OT-I/TCRwt or OT-I/TCRmyc were stained with a CD8-specific antibody, an OT-I-specific tetramer and an irrelevant tetramer. Cells shown are gated on CD8 expression. Untransduced splenocytes (neg) were used as a control (boxed in grey). Numbers indicate the MFI of the tetramer staining. (B) CD8α-positive 58 cells were transduced with the myc-tagged or wt OT-I/TCR, stained with vα2- and vβ5-specific antibodies and analyzed by FACS. Numbers show the percentage of transduction efficiency. (C) 1 x 10⁵ OT-I/TCRwt- or OT-I/TCRmyc-transduced 58 (CD8α+) cells were stimulated for 24 hours with 1 x 10⁵ T2-K^b cells pulsed with 100 µM to 100 pM ova peptide. As a control untransduced 58 cells were used (neg). IL-2 concentration of the culture supernatant was analyzed by ELISA. Unloaded T2-K^b cells (w/o) or T2-K^b cells loaded with irrelevant peptide (irr) served as negative target controls. Data represent mean values of duplicates and error bars indicate the SD.
• Figure 17 :   T c ells transduced with myc-tagged TCRs can be depleted in vivo . Splenocytes of B6 mice were transduced with OT-I/TCRmyc. 5 x 10⁶ TCR-transduced cells were adoptively transferred i.v. into Rag-1^-/- recipients. (A) After 13 days blood was stained for presence of CD8- and myc-positive cells. (B) One group of mice received 500 µg of a myc-specific antibody i.p. for depletion. One day after antibody injection blood samples were collected and analyzed again with CD8- and myc-specific antibodies. As a control, one group of mice received OT-I/TCRwt-transduced cells, which were stained with CD8-, vα2- and vβ5-specific antibodies, and anti-myc antibody treatment (boxed in grey). The stainings show cells gated on CD8 expression and are representative for one of two treated animals.
• Figure 18 : OT-I TCR-transduced T cells induce diabetes only in previously irradiated RIP-mOVA mice. B6 splenocytes were transduced with OT-I/TCRwt and 1.5 x 10⁷ or 1.5 x 10⁶ OT-I TCR-positive T cells were injected i.v. into RIP-mOVA mice (n=1) which had either been irradiated or treated with cyclophosphamide before adoptive transfer. (A) Seven days after transfer blood samples were stained with CD8-, vα2- and vβ5-specific antibodies and analyzed by FACS. As a control blood from mice, which had either been irradiated or received cyclophosphamide but were not injected with T cells, was tested. Blood from an untreated B6 mouse served as a control for the endogenous vα2- and vβ5-positive T cell population. Cells shown are gated on CD8 expression. (B) Blood glucose levels of all mice were determined and followed up to 30 days. The data of irradiated mice are depicted using filled symbols; that of mice treated with cyclophosphamide using open symbols.
• Figure 19 :   Auto-immune diabetes induced by OT-I/TCR-transduced T cells can only be cured by an early administration of antibody. B6 splenocytes were transduced with OT-I/TCRmyc and 1.5 x 10⁷ transduced cells were transferred into irradiated RIP-mOVA mice. One mouse did not receive T cells as a negative control. Animals of both groups “late myc-ab” and “early myc-ab” (each n=2) were injected on day 4 after transfer with 500 µg myc-specific antibody. Note that mice in the first one are glycemic at this time-point, whereas in the second one mice show normal blood glucose levels. Mice in group “late myc-ab (rep)” (n=2) received additionally the same dose of antibody on days 6, 10, and 13. As a control animals in group “no myc-ab” (n=2) were not treated with antibody. Blood glucose levels were determined and are depicted as mean values from animals in one group, SD is indicated by the error bars.
• Figure 20 :   Treatment of auto-immune insulitis mediated by myc-specific antibody depletion of OT-I/TCRmyc transduced auto-reactive T cells. B6 splenocytes were transduced with either OT-I/TCRwt or OT-I/TCRmyc retroviruses and 2 x 10⁷ TCR-positive cells were injected i.v. into sub-lethally irradiated RIP-mOVA mice. Mice which were irradiated but received no cells served as a negative control. 500 µg of a myc-specific antibody was administered i.p. into all mice that had received T cells harboring the TCRwt and half of the mice which had received T cells carrying the TCRmyc. Blood glucose concentration was determined. Depicted are mean values of all animals (n=5) in one group; error bars indicate SD.
• Figure 21 : Immunohisto logy shows T cell infiltration and destruction of islets in the pancreas. (A) Two days after adoptive transfer, pancrei of mice from each group were analyzed by IHC with ovalbumin- (red) and CD8- (green) specific antibodies. Nuclei were stained with DAPI (blue). (B) Pancreatic sections from diabetic and antibody-treated mice were stained on day six in the same way. Insets show larger parts of the tissue at a lower magnification.
• Figure 22 :   Depletion of OT-I/TCRmyc T cells can be detected in mesenterial lymph nodes and spleen.  Lymphocytes were isolated from (A) mesenterial lymph nodes and (B) spleen of animals from all groups and analyzed with a CD8-specific antibody and OT-I specific tetramer using flow cytometry. Lymphocytes from mice that did not receive T cells served as a negative control (boxed in grey). Depicted cells are gated on positive CD8 expression.
• Figure 23 : The human gp100/TCRmyc can be expressed comparable to gp100/TCRwt and is d e tected by a myc-specific antibody. (A) The TCR-deficient human T cell line Jurkat76 was transduced either with gp100/TCRwt or gp100/TCRmyc, enriched with vβ8-chain-specific antibodies and subcloned by limiting dilution. TCR expression was analyzed by flow cytometry staining with a vβ8-specific antibody. Untransduced cells (neg, boxed in grey) served as a negative control. (B) Jurkat76 cells transduced with gp100/TCRmyc were stained with a myc-specific antibody and analyzed by flow cytometry. Cells transduced with the unmodified wild-type receptor served as a control. The data show results of one representative clone of several that were tested.
• Figure 24: The human gp100/TCRmyc functions comparably to its TCRwt counterpart. (A) PBLs were transduced with gp100/TCRwt or gp100/TCRmyc retroviruses and stained with a vβ8-specific antibody and a gp100-specific tetramer. Untransduced PBLs (neg, boxed in grey) show the background of endogenous vβ8- positive T cells. Numbers indicate the MFI of the tetramer staining. (B) gp100/TCRwt- or gp100/TCRmyctransduced PBLs were co-cultured with T2 cells pulsed with 10 μM to 10 pM gp100 peptide for 24 hours. Untransduced PBLs were used as a negative control (neg). Culture supernatant was analyzed for IFN-γ content by ELISA. Unloaded T2 cells (w/o) or T2 cells loaded with an irrelevant NYeso1-derived peptide (irr) served as negative target controls. Data represent mean values of duplicates and error bars indicate SD. The results were reproduced in two independent experiments and with two different donors.
• Figure 25 :   T cells transduced with gp100/TCRmyc can be depleted in vitro by complement- and cell-mediated lysis. PBLs were transduced with gp100/TCRmyc retroviruses, sorted for myc-positive cells and restimulated with gp100 peptide-pulsed T2 cells. (A) For complement-mediated depletion, cells were incubated with a myc-specific antibody and rabbit complement factors. 7-AAD staining was used to discriminate between living and dead cells. Cells incubated without antibody served as a control. (B) For cell-mediated lysis, autologous PBMCs enriched for NK cells were used as effector cells. ⁵¹Chromium-labeled TCRmyc-positive PBLs were incubated with effector cells in E:T ratios from 50:1 to 2:1. A myc-specific antibody and a secondary rabbit anti-mouse IgG1 antibody were added and lysis was measured in a standard four-hour chromium release assay. Samples without antibody served as a control. Data represent mean values of duplicates and error bars indicate the SD. Similar results were obtained in an independent experiment with a different donor.