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
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

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

slide8
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
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
ad