Genetic manipulation of donor t cells to control graft versus host disease gvhd
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Genetic manipulation of donor T cells to control Graft versus Host disease (GvHD). Jordan Laboratory for hemato-oncology Hematology Department Eric Lammertsma - Tineke Lenstra - Hiljanne van der Meer Tom van Meerten – Samantha Hol. Contents. Introduction Approach Methods Conclusions

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Genetic manipulation of donor T cells to control Graft versus Host disease (GvHD)

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Genetic manipulation of donor t cells to control graft versus host disease gvhd

Genetic manipulation of donor T cells to control Graft versus Host disease (GvHD)

Jordan Laboratory for hemato-oncology Hematology Department

Eric Lammertsma - Tineke Lenstra - Hiljanne van der Meer

Tom van Meerten – Samantha Hol


Contents

Contents

  • Introduction

  • Approach

  • Methods

  • Conclusions

  • Discussion


Introduction

Introduction

  • Haematological malignancies

    -> Stem Cell Transplantation

    -> Complication: Graft versus Host Disease


Role of donor t cells in sct

Role of donor T-cells in SCT


Treatment of gvhd

Treatment of GvHD

  • Immunosuppressive drugs

  • Suicide gene therapy:

  • HSV-thymidine kinase with ganciclovir


Suicide gene therapy cd20 cd20

Suicide gene therapy: CD20/αCD20

  • Transduce T-cells with CD20-gene

  • Transplant

  • T-cell depleted Hematopoeitic Stem Cells

  • CD20 T-cells

  • GvHD -> αCD20 = Rituximab


Suicide gene therapy cd20 cd201

Suicide gene therapy: CD20/αCD20

Rituximab (CD20 antibody) + complement


Genetic manipulation of donor t cells to control graft versus host disease gvhd

FACS

SS

SS

FS

FS

Side Scatter (SSC)

Side Scatter (SSC)

Forward Scatter (FSC)

Forward Scatter (FSC)


Approach

Approach

  • Transfect Ph-ampho cells to produce virus with CD20 vector

  • Transduce CD20 DNA to human CEM cells

  • Examine the expression of CD20

  • Examine the effect of Rituximab on transduced cells


Transfection

Transfection

  • Ph-ampho cells for virus production

  • Gene variants:

  • CD20

  • CD20 wpre ic

  • CD20 wpre

  • CD20 ins9


Transfection1

Transfection

  • CD20, CD20 wpre ic and CD20 wpre transfected

  • CD20 ins9 not expressed

  • Virus harvested


Transduction

Transduction

  • CEM cells (human donor T cells)

  • Incubated with 4 virus concentrations

  • Examined CD20 expression

    (transduction efficiency)


Transduction1

Transduction

  • CD20 expression in transduced T cells


Transduction2

Transduction


Effect of rituximab

Effect of Rituximab

Rituximab (CD20 antibody) + complement


Effect of rituximab1

Effect of Rituximab

Clones

Control


Conclusions

Conclusions

  • CD20 T-cell production possible

  • Rituximab causes cell lysis ->

    independent of level of CD20 expression


Discussion

Discussion

  • No reliable negative control

  • Up to 10% CD20 T-cell survival

  • Rituximab specific for CD20 (?)

  • CD20 on human B-cells


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